Vanadium compounds for treating cancer

ABSTRACT

The invention provides methods for treating cancer and vanadium compounds that are useful for the treatment of tumors, as well as pharmaceutical compositions comprising the compounds, and synthetic methods and intermediates useful for preparing the compounds.

PRIORITY OF INVENTION

This application claims priority to U.S. Provisional application No.60/097,377, filed Aug. 21, 1998 U.S. Pat. No. 6,245,808 and is aContinution-in-part of U.S. application Ser. No. 09/187,115, filed Nov.5, 1998 and is a Continuation-in-part of U.S. application Ser. No.09/008,898, filed Jan. 20, 1998 U.S. Pat. No. 6,051,603.

FIELD OF THE INVENTION

The present invention relates to Vanadium (IV) compounds effective fortreating tumor cells and particularly effective to induce apoptosis inleukemia cells, breast cancer cells, prostate cancer cells, and braincancer cells.

BACKGROUND OF THE INVENTION

Cancer is a major disease that continues as one of the leading causes ofdeath at any age. In the United States alone, it is anticipated thatmore than half a million Americans will die of cancer in 1999.Currently, radiotherapy and chemotherapy are two important methods usedin the treatment of cancer.

Considerable efforts are underway to develop new chemotherapeutic agentsfor more potent and specific anti-cancer therapy, presenting effectiveand efficient cytotoxicity against tumor cells, with minimalinterference with normal cell function. Accordingly, there is an urgentneed for the development and analysis of novel, effective anti-canceragents.

A single vanadocene (IV) compound (e.g., VCp₂Cl₂) is reported as havingbiological activity.

Sakurai, et. al, BBRC, Vol. 206, p. 133 (1995) discloses an oxovanadiumcompound (e.g., [VO(Phen)(H₂O)₂](SO₄)) that is active againstpharyngonasal cancer as determined by a single assay.

Holmes, Ph.D. Thesis, LSU (1961) discloses oxovanadium compounds (e.g.,[VO(SO₄)(Phen)₂] and [VO(ClO₄)(Bpy)₂]) but does not disclose biologicaldata for the compounds.

Selbin, Chem. Rev., Vol. 65, p. 155 (1965) discloses oxovanadiumcompounds (e.g., [VO(SO₄)(Phen)₂] and [VO(ClO₄)(Bpy)₂]) but does notdisclose biological data for the compounds.

SUMMARY OF THE INVENTION

The invention provides a method for treating cancer in a mammalcomprising administering to the mammal in need of such treatment aneffective amount of a vanadium (IV) compound; or a pharmaceuticallyacceptable salt thereof; with the proviso that the vanadium (IV)compound is not VCp₂Cl₂ or [VO(Phen)(H₂O)₂](SO₄).

The invention also provides a method for treating a mammal inflictedwith cancer comprising administering to the mammal in need of suchtreatment an effective amount of an oxovanadium compound; or apharmaceutically acceptable salt thereof; with the proviso that thecancer is not pharyngonasal cancer.

The invention also provides a method for treating a mammal inflictedwith cancer comprising administering to the mammal in need of suchtreatment an effective amount of a vanadocene compound; or apharmaceutically acceptable salt thereof; with the proviso that thevanadocene compound is not VCp₂Cl₂.

The invention also provides a compound of formula II:

wherein R and R¹ are each independently H, (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro; Xand X¹ are each independently a monodentate or bidentate ligand, or noligand is present on X¹; and Y is a monodentate ligand; or apharmaceutically acceptable salt thereof; with the proviso that thecompound is not [VO(SO₄)(Bpy)₂].

The invention also provides a compound of formula III:

wherein R² and R³ are each independently H, (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro; X²and X³ are each independently a monodentate or bidentate ligand, or noligand is present on X³; and Z is O, CH₂, CH₂—CH₂ or CH═CH; or apharmaceutically acceptable salt thereof; with the proviso that thecompound is not [VO(Phen)(H₂O)₂](SO₄) or [VO(SO₄)(Phen)₂].

The invention also provides a compound of formula IV:

wherein R⁴, R⁵ and R⁶ are each independently H, (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro; X⁴and X⁵ are each independently a monodentate or bidentate ligand, or noligand is present on X⁵; Y is a monodentate ligand, or no ligand ispresent on X⁵; and Y is a monodentate ligand; or a pharmaceuticallyacceptable salt thereof; with the proviso that the compound is not[VO(Phen)(H₂O)₂](SO₄) or [VO(SO₄)(Phen)₂].

The invention also provides a compound of formula V:

wherein R⁷ and R⁹ are each independently H, (C1-C3)alkyl, (C1-C3)alkoxy,or halo(C1-C3)alkyl; R⁸ is H, (C1-C3)alkyl, halo, (C1-C3)alkoxy, orhalo(C1-C3)alkyl; Y and Y¹ are each independently a monodentate orbidentate ligand; and n is 0 or 1; or a pharmaceutically acceptable saltthereof.

The invention also provides a compound of formula VII:

wherein R¹ and R² are each independently a cyclopentadienyl ring,wherein any cyclopentadienyl ring may optionally be substituted with oneor more (C₁-C₃)alkyl; and R¹⁰-R¹³ are each independently H, halo, or(C1-C6)alkyl; or a pharmaceutically acceptable salt thereof.

The invention also provides a pharmaceutical composition comprising acompound of formula II:

wherein R and R¹ are each independently H, (C₁-C₃)alkyl, halogen,(C₁-C₃)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro; Xand are each independently a monodentate or bidentate ligand; or noligand is present on X¹; and Y is a monodentate ligand, or no ligand ispresent on X¹; and Y is a monodentate ligand or a pharmaceuticallyacceptable salt thereof; and a pharmaceutically acceptable carrier.

The invention also provides a pharmaceutical composition comprising acompound of formula III:

wherein R² and R³ are each independently H, (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro; X²and X³ are each independently a monodentate or bidentate ligand, or noligand is present on X³; and Z is O, CH₂, CH₂—CH₂ or CH═CH; or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier; with the proviso that the compound is not[VO(Phen)(H₂O)₂](SO₄).

The invention also provides a pharmaceutical composition comprising acompound of formula IV:

wherein R⁴, R⁵ and R⁶ are each independently H, (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro; X⁴and X⁵ are each independently a monodentate or bidentate ligand, or noligand is present on X⁵; Y is a monodentate ligand; or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier; with the proviso that the compound is not[VO(Phen)(H₂O)₂](SO₄).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Illustrates cytotoxic activity of vanadocenes on humantesticular cancer cell lines with (A) Tera-2 cells and (B) Ntera-2cells. Cells were incubated with increasing concentrations (1.9 μM-250μM) of 5 representative vanadocenes, VDOCN, VDSCN, VDSeCN, VDT, andVD(dtc) in DMSO for 24 hr in 96-well plates and the cell survival wasdetermined by the MTT assay as described in materials andmethodsActivity is expressed relative to DMSO controls. The data pointsrepresent the mean value of triplicates. The SD for each compound was<5% of the mean values.

FIG. 2. Illustrates VDSeCN and VDSCN induce apoptosis in humantesticular cancer cells. (Left panels) TUNEL analysis: Two-color flowcytometric contour plots of Ntera-2 cells treated with and withoutvanadocenes. Cells were incubated for 24 h in either control medium(0.1% DMSO) [A], or in medium supplemented with 100 μM VDSCN [B] orVDSeCN [C] in 0.1% DMSO, fixed, permeabilized, and visualized forDNA-fragmentation in a TUNEL assay using TdT and FITC-dUTP. Redfluorescence represents nuclei counterstained with propidium iodide.Percentages indicate cells with increased dUTP incorporation. (Rightpanels) Confocal images: Two-color confocal laser scanning microscopyimages of control and apoptotic cells. B) Control cells visualized fordUTP incorporation using FITC-dUTP. D and F) Apoptotic nuclei of cellstreated with VDSCN and VDSeCN are recognized by fluorescein labeledgreen/yellow (superimposed red plus green) fluorescence. Originalmagnification×600.

FIG. 3. Illustrates vanadocenes induce apoptosis in human testicularcancer cells. [Left panels] FACS analysis: Two-color flow cytometriccontour plots of Tera-2 cells treated with and without vanadocenes.Cells were incubated for 24 h in either control medium (0.1% DMSO) (A),or in medium supplemented with 100 μM VDA (C), VDCN (E), VDOCN (G), orVDCO (I) in 0.1% DMSO, fixed, permeabilized, and visualized forDNA-fragmentation in a TUNEL assay using TdT and FITC-dUTP. Redfluorescence represents nuclei counterstained with propidium iodide.Percentages indicate cells with increased dUTP incorporation. [Rightpanels] Confocal images: Two-color confocal laser scanning microscopyimages of control and apoptotic cells. (B) Control cells visualized fordUTP incorporation using FITC-dUTP. Apoptotic nuclei of cells treatedwith VDA (D), VDCN (F), VDOCN (H) and VDCO (J) are recognized byfluorescein labeled green/yellow (superimposed red plus green)fluorescence. Original magnification×600 are recognized by fluoresceinlabeled (green or yellow[i.e., superimposed red plus green] color)nuclei and apoptotic bodies within the nuclei. (originalmagnification×600).

FIG. 4. Illustrates the formation of hyperdiploid nuclei and inductionof apoptosis in vanadocene-treated Tera-2 testicular cancer cells. Cellswere treated with vehicle, 12.5, 25, or 50 μM venadocene for 24 h,stained with propidium iodide and analysed by flow cytometry for DNAcontent. The percentages indicate the hyperdiploid/apoptotic nuclei.

FIG. 5 Illustrates Cytotoxic activity of metallocene dichlorides (A) andvanadocene on human glioblastoma cells. U373 glioblastoma cells wereincubated with increasing concentrations of 5 metallocene dichloridesVDC, TDC, ZDC, HDC, and MDCT or 7 representative vanadocene VDC, VDSeCN,VDI, VDA, VDCN, VDH, and VDB for 24 hr in 96-well plates and the cellsurvival was determined by the MTT assay as described in materials andmethods. Activity is expressed relative to DMSO controls. The datapoints represent the mean (±SD) value of three independent experiments.

FIG. 6. Illustrates the cytotoxic activity of VDSeCN in 4 human cancercell lines. Fluorescence-activated cell sorter-correlated two parameterdisplay (fluorescence from propidium iodide [PI], and fluorescence fromMC540 staining) of AML (HL-60), breast cancer (MDA-MB-231 and BT-20) andbrain tumor (U87) cells stained with MC540 and PI 24 h after treatmentwith vehicle (CON), 10, 50, or 100 μM of VDSeCN. The percentagesindicate the fraction of cells at an early stage of apoptosis, asmeasured by single MC540 fluorescence, and the fraction of cells at anadvanced stage apoptosis, as measured by dual MC540/PI fluorescence.

FIG. 7. Illustrates the formation of hyperdiploid nuclei and inductionof apoptosis in vanadocene-treated AML HL-60 cells. Cells were treatedwith vehicle (Con 1 and Con 2), 10, 50 and 100 μM VDC or VDSeCN for 24h, stained with propidium iodide and analysed by flow cytometry for DNAcontent. The percentages indicate the hyperdiploid/apoptotic nuclei.

FIG. 8. Illustrates the morphological features of breast cancer BT-20cells treated with VDC. cells were incubated with vehicle (A), or 25 μMof VDC for 48 hr (B) or 72 hr (C) 24 hr and processed forimmunofluorescence using a monoclonal antibody to α-tubulin (greenfluorescence). VDC-treated cells showed marked shrinkage with disruptionof microtubules and lost their ability to adhere to the substratum. Bluefluorescence represents nuclei stained with TOTO-3.

DETAILED DESCRIPTION

Vanadium is a physiologically essential element that can be found inboth anionic and cationic forms with oxidation states ranging from −3 to+5 (I-V). This versatility provides unique properties to vanadiumcomplexes. In particular, the catonic form of vanadium complexes withoxidation state +4 (IV) have been shown to function as modulators ofcellular redox potential, regulate enzymatic phosphorylation, and exertpleiotropic effects in multiple biological systems by catalyzing thegeneration of reactive oxygen species (ROS). Besides the ability ofvanadium metal to assume various oxidation states, its coordinationchemistry also plays a key role in its interactions with variousbiomolecules. In particular, organometallic complexes of vanadium (IV)linked to bis (cycopentadienyl) moieties or vanadocene derivativesexhibit antitumor properties both in vitro and in vivo primarily viaoxidative damage.

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote both straightand branched groups; but reference to an individual radical such as“propyl” embraces only the straight chain radical, a branched chainisomer such as “isopropyl” being specifically referred to.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine antitumor activity using thestandard tests described herein, or using other similar tests which arewell known in the art.

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C₁-C₃)alkylcan be methyl, ethyl or propyl; halo(C1-C3)alkyl can be iodomethyl,bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl,2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; (C1-C3)alkoxycan be methoxy, ethoxy, or propoxy; and (C₂-C₆)alkanoyloxy can beacetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, orhexanoyloxy.

As used herein, the following definitions define the stated terms:

“Organometallic compound” is an organic compound comprised of a metalattached directly to carbon (R-M).

“Coordination compound” is a compound formed by the union of a centralmetal atom or ion with ions or molecules called ligands or complexingagents.

“Ligand” or a “complexing agent” is a molecule, ion or atom that isattached to the central metal atom or ion of a coordination compound.

“Monodentate ligand” is a ligand having a single donor atom coordinatedto the central metal atom or ion.

“Bidentate ligand” is a ligand having two donor atoms coordinated to thesame central metal atom or ion.

“Oxovandium (IV) complex” is a coordination compound including vanadiumas the central metal atom or ion, and the vanadium has an oxidationstate of +4 (IV), and is double bonded to oxygen.

The present invention concerns organometallic vanadium complexes, andthe finding that such oxovanadium complexes have potent and selectiveantitumor activity, and are particularly active and stable antitumoragents.

Compounds of the invention include oxovanadium (IV) containingorganometallic complexes having antitumor activity. Preferred theoxovanadium (IV) complexes include at least one bidentate ligand.Suitable bidentate ligands include N, N; N, O; and O, O bidentateligands. Examples of suitable bidentate ligands include bipyridyl,bridged bipyridyl, and acetophenone. Particularly, preferred oxovanadiumcompounds of the invention are those having the formulas II, III, IV, VIand VIII shown and described below.

Specifically, the vanadium (IV) compound is a compound of formula I:

wherein,

R₁-R₂ are each independently halo, OH₂, O₃SCF₃, N₃, CN, OCN, SCN orSeCN;

R₃-R₄ are each independently a cyclopentadienyl ring, wherein eachcyclopentadienyl ring is optionally substituted with one or more(C1-C3)alkyl.

Specifically, halo is chloro, bromo, or iodo.

Specifically, (C1-C3)alkyl is methyl. Specifically, the compound offormula I is VCp₂Cl₂, VCp₂Br₂, VCp₂I₂, VCp₂X₂, VCp₂(N₃)₂, VCp₂(CN)₂,VCp₂(NCO)₂, VCp₂(NCO)Cl, VCp₂(NCS)₂. 0.5H₂O, VCp₂(NCSe)₂,VCp₂Cl(CH₃CN)(FeCl₄), VCp₂(O₃SCF₃)₂, V(MeCp)₂ Cl₂. 0.5 H₂O,V(Me₅Cp)₂Cl₂, VCp₂(acac), VCp₂(hf-acac), VCp₂(bpy), VCp₂(cat),VCp₂(dtc), VCp₂PH, or VCp₂H.

Specifically, the vanadium (IV) compound is a compound of formula II:

wherein

R and R¹ are each independently H, (C1-C3)alkyl, halogen, (C1-C3)alkoxy,halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro;

X and X¹ are each independently monodentate or bidentate ligands, or noligand is present on X¹;

Y is a monodentate ligand; and

Specifically, X and X¹ are each independently OH₂, a bidentate ligand,or a monodentate ligand; wherein each ligand is optionally substitutedwith one or more (C1-C3)alkyl.

Specifically, (C1-C3)alkyl is methyl.

Specifically, R and R¹ are each independently H or (C1-C3)alkyl.

Specifically, the compound of formula II is [VO(Bpy)(H₂O)₂](SO₄),[VO(SO₄)(Bpy)₂], [VO(Me₂-bpy)(H₂O)₂](SO₄), or [VO(SO₄)(Me₂-bpy)₂].

Specifically, the vanadium (IV) compound is a compound of formula III:

wherein

R² and R³ are each independently H, (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro;

X² and X³ are each independently monodentate or bidentate ligands, or noligand is present on X³;

Z is selected from O, CH₂, CH₂—CH₂ and CH═CH; and

Specifically, Z is CH═CH.

Specifically, Y is OH₂ or OSO₃.

Specifically, X² is OH₂, a bidentate ligand, or a monodentate ligand.

Specifically, X³ is a bidentate ligand or a monodentate ligand.

Specifically, R² and R³ are each independently H, (C1-C3)alkyl, halo, ornitro.

Specifically, (C1-C3)alkyl is methyl.

Specifically, halo is chloro.

Specifically, the compound of formula III is [VO(Bpy)(H₂O)₂](SO₄),[VO(SO₄)(Bpy)₂], [VO(Me₂-bpy)(H₂O)₂](SO₄), [VO(SO₄)(Me₂-bpy)₂],[VO(Phen)(H₂O)₂](SO₄), [VO(SO₄)(Phen)₂], [VO(Me₂-Phen)(H₂O)₂](SO₄), or[VO(SO₄)(Me₂-Phen)₂].

Specifically, the vanadium (IV) compound is a compound of formula IV:

wherein

R⁴, R⁵ and R⁶ are each independently H, (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro;

X⁴ and X⁵ are each independently a monodentate or bidentate ligand, orno ligand is present on X⁵;

Y is a monodentate ligand; and

Specifically, R⁴-R⁶ are each independently H, (C1-C3)alkyl, halo ornitro.

Specifically, (C1-C3)alkyl is methyl.

Specifically, halo is chloro.

Specifically, Y is OH₂ or OSO₃,.

Specifically, X⁵ is OH₂, a bidentate ligand, or a monodentate ligand.

Specifically, the compound of formula IV is [VO(Bpy)(H₂O)₂](SO₄),[VO(SO₄)(Bpy)₂], [VO(Me₂-bpy)(H₂O)₂](SO₄), [VO(SO₄)(Me₂-bpy)₂],[VO(Phen)(H₂O)₂](SO₄), [VO(SO₄)(Phen)₂], [VO(Me₂-Phen)(H₂O)₂](SO₄),[VO(SO₄)(Me₂-Phen)₂], [VO(Cl-Phen)(H₂O)₂](SO₄), [VO(SO₄)(Cl-Phen)₂],[VO(NO₂-Phen)(H₂O)₂](SO₄), or [VO(SO₄)(NO₂-Phen)₂].

Specifically, the cancer is testicular cancer, Hodgkin's lymphoma,multiple myeloma, or non-Hodgkin's lymphoma.

Another specific method of the invention comprises administering anoxovanadium compound.

Another specific method of the invention comprises administering avanadocene compound with the proviso that the vanadocene compound is notVCp₂Cl₂.

The invention provides novel compounds. Accordingly, there is provided acompound of formula II:

wherein

R and R¹ are each independently H, (C1-C3)alkyl, halogen, (C1-C3)alkoxy,halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro;

X and X¹ are each independently a monodentate or bidentate ligand, or noligand is present on X¹; and

Y is a monodentate ligand;

or a pharmaceutically acceptable salt thereof;

with the proviso that the compound is not [VO(SO₄)(Bpy)₂].

Specifically, X and X¹ are each independently OH₂, a bidentate ligand,or a monodentate ligand; wherein each ligand is optionally substitutedwith one or more (C1-C3)alkyl.

Specifically, (C1-C3)alkyl is methyl.

Specifically, R and R¹ are each independently H or (C1-C3)alkyl.

Specifically, the compound of formula II is [VO(Bpy)(H₂O)₂](SO₄),[VO(SO₄)(Bpy)₂], [VO(Me₂-bpy)(H₂O)₂](SO₄), or [VO(SO₄)(Me₂-bpy)₂].

Another specific compound of the present invention is a compound offormula III:

wherein

R² and R³ are each independently H, (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro;

X² and X³ are each independently a monodentate or bidentate ligand, orno ligand is present on X³; and

Z is O, CH₂, CH₂—CH₂ or CH═CH;

or a pharmaceutically acceptable salt thereof;

with the proviso that the compound is not [VO(Phen)(H₂O)₂](SO₄) or[VO(SO₄)(Phen)₂].

Specifically, Z is CH═CH.

Specifically, Y is OH₂ or OSO₃.

Specifically, X² is OH₂, a bidentate ligand, or a monodentate ligand.

Specifically, X³ is a bidentate ligand or a monodentate ligand.

Specifically, R² and R³ are each independently H, (C1-C3)alkyl, halo, ornitro.

Specifically, (C1-C3)alkyl is methyl.

Specifically, halo is chloro.

Specifically, the compound of formula III is [VO(Bpy)(H₂O)₂](SO₄),[VO(SO₄)(Bpy)₂], [VO(Me₂-bpy)(H₂O)₂](SO₄), [VO(SO₄)(Me₂-bpy)₂],[VO(Phen)(H₂O)₂](SO₄), [VO(SO₄)(Phen)₂], [VO(Me₂-Phen)(H₂O)₂](SO₄), or[VO(SO₄)(Me₂-Phen)₂].

Another specific compound of the present invention is a compound offormula IV:

wherein

R⁴, R⁵ and R⁶ are each independently H, (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro;

X⁴ and X⁵ are each independently a monodentate or bidentate ligand; and

Y is a monodentate ligand

or a pharmaceutically acceptable salt thereof;

with the proviso that the compound is not [VO(Phen)(H₂O)₂](SO₄) or[VO(SO₄)(Phen)₂].

Specifically, R⁴-R⁶ are each independently H, (C1-C3)alkyl, halo ornitro.

Specifically, (C1-C3)alkyl is methyl.

Specifically, halo is chloro.

Specifically, Y is OH₂ or OSO₃,.

Specifically, X⁵ is OH₂, a bidentate ligand, or a monodentate ligand.

Specifically, the compound of formula IV is [VO(Bpy)(H₂O)₂](SO₄),[VO(SO₄)(Bpy)₂], [VO(Me₂-bpy)(H₂O)₂](SO₄), [VO(SO₄)(Me₂-bpy)₂],[VO(Phen)(H₂O)₂](SO₄), [VO(SO₄)(Phen)₂], [VO(Me₂-Phen)(H₂O)₂](SO₄),[VO(SO₄)(Me₂-Phen)₂], [VO(Cl-Phen)(H₂O)₂](SO₄), [VO(SO₄)(Cl-Phen)₂],[VO(NO₂-Phen)(H₂O)₂](SO₄), or [VO(SO₄)(NO₂-Phen)₂].

Another specific compound of the present invention is a compound offormula V:

wherein

R⁷ and R⁹ are each independently H, (C1-C3)alkyl, (C1-C3)alkoxy, orhalo(C1-C3)alkyl;

R⁸ is H, (C1-C3)alkyl, halo, (C1-C3)alkoxy, or halo(C1-C3)alkyl;

Y and Y¹ are each independently a monodentate or bidentate ligand; and

n is 0 or 1;

or a pharmaceutically acceptable salt thereof.

Another specific compound of the present invention is a compound offormula VII:

wherein

R¹ and R² are each independently a cyclopentadienyl ring, wherein anycyclopentadienyl ring may optionally be substituted with one or more(C1-C6)alkyl; and

R¹⁰-R¹³ are each independently H, halo, or (C1-C6)alkyl;

or a pharmaceutically acceptable salt thereof.

Specifically, R¹ and R² are each a cyclopentadienyl ring.

Specifically, R¹⁰-R¹³ are each H.

Specifically, the compound of formula VII is the compound Cp₂V(O₂C₆H₄).

A specific pharmaceutical composition of the present invention comprisesa compound of formula II:

wherein

R and R¹ are each independently H, (C1-C3)alkyl, halogen, (C1-C3)alkoxy,halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro;

X and are each independently a monodentate or bidentate ligand, or noligand is present on X¹; and

Y is a monodentate ligand;

or a pharmaceutically acceptable salt thereof;

and a pharmaceutically acceptable carrier.

Specifically, X and X¹ are each independently OH₂, a bidentate ligand,or a monodentate ligand, wherein each ligand is optionally substitutedwith one or more (C1-C3)alkyl.

Specifically, (C1-C3)alkyl is methyl.

Specifically, R and R¹ are each independently H or (C1-C3)alkyl.

Specifically, the compound of formula II is [VO(Bpy)(H₂O)₂](SO₄),[VO(SO₄)(Bpy)₂], [VO(Me₂-bpy)(H₂O)₂](SO₄), or [VO(SO₄)(Me₂-bpy)₂].

Another specific pharmaceutical composition of the present inventioncomprises a compound of formula III:

wherein

R² and R³ are each independently H, (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro;

X² and X³ are each independently a monodentate or bidentate ligand, orno ligand is present on X³; and

Z is O, CH₂, CH₂—CH₂ or CH═CH;

or a pharmaceutically acceptable salt thereof;

and a pharmaceutically acceptable carrier;

with the proviso that the compound is not [VO(Phen)(H₂O)₂](SO₄).

Specifically, Z is CH═CH.

Specifically, Y is OH₂ or OSO₃.

Specifically, X² is OH₂, a bidentate ligand, or a monodentate ligand.

Specifically, X³ is a bidentate ligand or a monodentate ligand.

Specifically, R² and R³ are each independently H, (C1-C3)alkyl, halo, ornitro.

Specifically, (C1-C3)alkyl is methyl.

Specifically, halo is chloro.

Specifically, the compound of formula III is [VO(Bpy)(H₂O)₂](SO₄),[VO(SO₄)(Bpy)₂], [VO(Me₂-bpy)(H₂O)₂](SO₄), [VO(SO₄)(Me₂-bpy)₂],[VO(Phen)(H₂O)₂](SO₄), [VO(SO₄)(Phen)₂], [VO(Me₂-Phen)(H₂O)₂](SO₄), or[VO(SO₄)(Me₂-Phen)₂].

Another pharmaceutical composition of the present invention comprises acompound of formula IV:

wherein

R⁴, R⁵ and R⁶ are each independently H, (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C₂-C₆)alkanoyloxy or nitro;

X⁴ and X⁵ are each independently a monodentate or bidentate ligand; and

Y is a monodentate ligand;

or a pharmaceutically acceptable salt thereof;

and a pharmaceutically acceptable carrier;

with the proviso that the compound is not [VO(Phen)(H₂O)₂](SO₄).

Specifically, R⁴-R⁶ are each independently H, (C1-C3)alkyl, halo ornitro.

Specifically, (C1-C3)alkyl is methyl.

Specifically, halo is chloro.

Specifically, Y is OH₂ or OSO₃,.

Specifically, X⁵ is OH₂, a bidentate ligand, or a monodentate ligand.

Specifically, the compound of formula IV is [VO(Bpy)(H₂O)₂](SO₄),[VO(SO₄)(Bpy)₂], [VO(Me₂-bpy)(H₂O)₂](SO₄), [VO(SO₄)(Me₂-bpy)₂],[VO(Phen)(H₂O)₂](SO₄), [VO(SO₄)(Phen)₂], [VO(Me₂-Phen)(H₂O)₂](SO₄),[VO(SO₄)(Me₂-Phen)₂], [VO(Cl-Phen)(H₂O)₂](SO₄), [VO(SO₄)(Cl-Phen)₂],[VO(NO₂-Phen)(H₂O)₂](SO₄), or [VO(SO₄)(NO₂-Phen)₂].

Administration of the compounds as salts may be appropriate. Examples ofacceptable salts include alkali metal (for example, sodium, potassium orlithium) or alkaline earth metal (for example calcium) salts, however,any salt that is non-toxic and effective when administered to the animalbeing treated is acceptable.

Acceptable salts may be obtained using standard procedures well known inthe art, for example by reacting a sufficiently acidic compound with asuitable base affording a physiologically acceptable anion.

The compositions of the invention can be formulated as pharmaceuticalcompositions and administered to an animal host, such as a human patientin a variety of forms adapted to the chosen route of administration,i.e., orally or parenterally, by intravenous, intramuscular, topical orsubcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained. When administered orally, the compositions of the inventioncan preferably be administered in a gelatin capsule.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The compositions of the invention may also be administered intravenouslyor intraperitoneally by infusion or injection. Solutions of the activecomposition can be prepared in water, optionally mixed with a nontoxicsurfactant. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, triacetin, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecomposition in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, the present compositions may be applied inpure form, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula I to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the present invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949.

Generally, the concentration of the compositions of the invention in aliquid composition, such as a lotion, will be from about 0.1-50 wt-%,preferably from about 0.5-5 wt %. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

The amount of the composition required for use in treatment will varynot only with the particular salt selected but also with the route ofadministration, the nature of the condition being treated and the ageand condition of the patient and will be ultimately at the discretion ofthe attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about0.1 to about 150 mg/kg, e.g., from about 10 to about 75 mg/kg of bodyweight per day, such as 3 to about 50 mg per kilogram body weight of therecipient per day, preferably in the range of 1 to 100 mg/kg/day, mostpreferably in the range of 5 to 20 mg/kg/day.

The compositions are conveniently administered in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.5 to about75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about30 μM. This may be achieved, for example, by the intravenous injectionof a 0.05 to 5% solution of the active ingredient, optionally in saline,or orally administered as a bolus containing about 1-100 mg of theactive ingredient. Desirable blood levels may be maintained bycontinuous infusion to provide about 0.01-5.0 mg/kg/hr or byintermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

The compositions of the invention are useful for prevention and ofcancer.

Following i.m. administration, the compositions of the invention enterthe blood stream within about 10-15 minutes and reach a maximumconcentration in the blood within one hour of administration, at whichpoint they can be found throughout the circulatory related organs.

The antitumor activity of the compositions of the invention can bedetermined using assays that are known in the art, or can be determinedusing assays similar to those described in the following examples.

EXAMPLES Abbreviations

Cp⁻, cyclopentadienyl anion; acac, acetonylacetonate; Bpy, 2,2′Bipyridine; Hfacac, hexafluoroacetylacetonate; Cat, catecholate; Dtc,diethyl dithio carbamate; PH, N-phenyl bezohydroxamic acids; H,acethydroxamic acid; OTf, trifluoromethane sulphonate; THF,tetrahydrofuran; DMSO, dimethyl sulfoxide; CH₃CN, acetonitrile; CH₂Cl₂,dichloromethane; d—d, laportte spin forbidden transitions; LMCT, ligandto metal charge transfer transitions; p-p*, intraligand charge transfertransitions; s, m, w: strong, medium, and weak; b, broad; v, very; sh,shoulder.

Materials and Methods

Reagents used were reagent grade, unless otherwise stated. All solventswere used as received from Aldrich Sure Seal bottle, <0.005% water.Tetrahydrofuran was dried by distillation over solid sodium.Dichloromethane (reagent grade) was purified as follows: stirredovernight with concentrated sulfuric acids, seperated, washed withsaturated aqueous NaHCO₃, washed with aqueous KOH/KCl, washed withdistilled water, dried over anhydrous MgSO₄, and distilled from KOH.Perrin, D. D., Armereago, W. L. F. In Purification of LaboratoryChemicals, 3rd.ed.; pergamon press: New York, 1988. Sodium thiophenolatewas prepared by the reaction of a stoichiometric amount of NaOMe withthiophenol in methanol. The precipitated solid was washed with coldmethanol and dried under high vacuum. All the solvents were deoxygenetedby purging with argon, and reactions were carried out under an argonatmosphere by using standard Schlenk techniques.

The infra-red spectral data were recorded on a FT-Nicolet model Protege460. The solid samples were taken in a KBr pellet and the frequencieswere generally in the range of 4000-500 cm⁻¹. UV-vis spectra wererecorded in a quartz cell or cuvette on Beckman model DU 7400spectrophotometer and the spectral band are registered between 250-800run range. NMR spectra were recorded in CDCl₃ or Me₂SO-d⁶ on a Varian(300 Mhz) NMR spectrometer. Chemical shifts are reported as δ valuesdownfield from an internal standard of Me₄Si. Melting points weredetermined with Melt-temp laboratory devices Inc. apparatus, attached toFluke 51 K/J Thermometer.

All elemental analysis were performed by Atlantic Microlab, Inc.,Norcross, Ga., and the analytical results are supplied as supportinginformation. unless otherwise stated all operations were carried out atroom temperature.

Example I Vanadocene Compounds

Chemical Synthesis

All the metal tetrachlorides, MCl₄ (M=Ti, V, Mo, Hf, & Zr) werepurchased from Aldrich Chemical Co. (Milwaukee, Wis.).

Compounds 1-25 are shown in Synthetic Schemes 1-4 and Tables 1-4.VCp₂Cl₂, TiCp₂Cl₂, ZrCp₂Cl₂, and MoCp₂Cl₂ were prepared by knownprocedures. Wilkinson, G., Birmingham, J. M. Bis-cyclopentadienylcompounds of Ti, Zr, V, Nb, and Ta. J. Am. Chem. Soc., 76: 4281-4284,1954. Cardin, D. J., Lappart, M. F., Raston, C. L., Riley, P. I. InComprehensive organometallic chemistry; Wilkinson, G. ed. New York:Pergamon; 3: 554-646, 1982. Eisch, J. J., King, R. B. Organometallicsynthesis, Academic press, New York, N.Y. Vol.1, 75-76, 1965. The puritywas checked by ¹HNMR and IR spectroscopy and by elemental analysis. TheHfCp₂Cl₂ was directly purchased from aldrich Chemical Co. The complexVCp₂Cl₂ was purified by anaerobic Soxhlet extraction with CH₂Cl₂ at 44°C. (under partial vacuum). TiCp₂Cl₂ was recrystallized from THF.

Compounds 1-5 may be prepared as shown in Synthetic Scheme 1; compounds6-15 may be prepared as shown in Synthetic Scheme 2; compounds 16-22 maybe prepared as shown in Synthetic Scheme 3; and compounds 23-25 may beprepared as shown in Synthetic Scheme 4 (below).

HfCp₂Cl₂, (Compound 1). Yield: 75%. M.P. 330-335 C. Anal. Calcd. forHfC₁₀H₁₀Cl₂: C, 31.62, H, 2.63; Cl, 18.71. Found: C, 32.09; H, 2.75; Cl,18.98. UV-vis: (CH₂Cl₂) λ_(max): 312(LMCT), 268, 232 (π-π*) nm. IR (KBrDisc): 3105(vs), 1365(m), 1126(m), 1014(vs), 920(s,d), 802(vs), 816(vs),611(m) cm⁻. ¹HNMR (δ ppm, CDCl₃): 6.37 (s, 10H, 2×C₅H₅).

MoCp₂Cl₂, (Compound 2). Yield: 37%. M.P. 220° C. Anal. Calcd. forMoC₁₀H₁₀Cl₂: C, 40.4, H, 3.37; Cl, 23.9. Found: C, 40.8; H, 3.39; Cl,24.4. UV-vis: (DMSO) λ_(max): 678, 436 (d—d), 299(LMCT), 283, 267 (π-π*)nm. IR (KBr Disc): 3093(vs), 1420(vs), 1375(m), 1100(m), 1060(m),825(vs,d), 590(m) cm⁻¹. ¹HNMR (δ ppm, DMSO-d⁶): 6.27 (s, 10H, 2×C₅H₅).

TiCp₂Cl₂, (Compound 3). Yield: 45%. M.P. 290° C. (decomposes). Anal.Calcd. for TiC₁₀H₁₀Cl₂: C, 48.2, H, 4.01; Cl, 28.5. Found: C, 48.56; H,4.03; Cl, 28.78. UV-vis: (CH₂Cl₂) λ_(max): 526, 391 (LMCT), 314, 255(π-π*) nm. IR (KBr Disc): 3105(m), 1441(s), 1368(m), 1130(m), 1016(vs),956(m), 872(s), 820(vs)) cm⁻¹. ¹HNMR (δ ppm, CDCl₃): (s, 10H, 2×C₅H₅).

ZrCp₂Cl₂, (Compound 4). Yield: 78%. M.P. 240-245° C. (decomposes). Anal.Calcd. for ZrC₁₀H₁₀Cl₂: C, 41.09, H, 3.4; Cl, 24.31. Found: C, 41.01; H,3.4; Cl, 24.84. UV-vis: (CH₂Cl₂) λ_(max): 341 (LMCT), 294, 236 (π-π*)nm. IR (KBr Disc): 3104(m), 1435(s), 1363(m), 1122(m), 1014(vs), 815(s),850(sb), 610(m)) cm⁻. ¹HNMR (δ ppm, CDCl₃): 6.46 (s, 10H, 2×C₅H₅).

VCp₂Cl₂, (Compound 5). Yield: 55%. M.P. 248-255° C. (decomposes). Anal.Calcd. for VC₁₀H₁₀Cl₂: C, 47.62, H, 3.97; Cl, 28.1. Found: C, 47.88; H,4.04; Cl, 27.64. UV-vis: (CH₂Cl₂) λ_(max): 776, 647 (d—d), 380 (LMCT),283, 244 (π-π*) nm. IR (KBr Disc): 3095(s), 1444(s), 1433(s), 1368(m),1363(m), 1130(m), 1070(m), 1010(m), 887(m), 825(vs) cm⁻¹.

VCp₂Br₂, (Compound 6). To a 20 ml acetone solution of VCp₂Cl₂(0.2 g, 8mmol) was added 0.7 g (80 mmol) of solid LiBr with stirring, and thereaction mixture was allowed to reflux for 4 h. The solvent was removedafterwards through vacuum and dried. The deep green product wasextracted with 50 ml of boiling CHCl₃ and the solution was saturatedwith dry HBr gas before it was left overnight for crystallization at−20° C. The bright green crystals were collected on a frit and washedwith hexane and diethyl ether. Yield: 90%. M.P. turns darker graduallyover the range 250-350° C. (decomposes). Anal. Calcd. for VC₁₀H₁₀Br₂: C,35.19, H, 2.29; Br, 46.92. Found: C, 35.19; H, 2.9; Br, 46.91. UV-vis:(CH₂Cl₂) λ_(max): 773, (d—d), 412 (LMCT), 298, 232 (π-π*) nm. IR (KBrDisc): 3089(vs), 1425(s), 1431(m), 1373(m), 1363(w), 1128(w), 1024(m),1014(m), 887(m), 825(vs) cm⁻¹.

VCp₂l₂, (Compound 7). To anhydrous THF (25 mL) were added VCp₂Cl₂ (0.15g, 6 mmol) and KI (0.99 g, 60 mmol). This reaction mixture was refluxedovernight under argon. The resulting dark red-brown solution wasseperated from the inorganic salts by filtration and the solvent wasevaporated under vacuum. The dark red materials were scratched out fromthe bottom of the container in the presence of dry hexane. The solventwas removed by cannula techniques with double edged needles under argonpressure. The solid was dried under vacuum and stored under argon. Thiscompound is extremely sensitive to moisture and readily decomposes inhalogenated solvents, but it is stable in DMSO. Yield: 55%. M.P.: Couldnot be measured; compound gets sticky during handling in air.Anal.Calcd.for VC₁₀H₁₀I₂: C, 27.58; H, 2.3; I, 58.4. Found: C, 28.1; H,2.42; I, 58.9 UV-Vis:(CH₂Cl₂) λ_(max): 620, 552, (d—d), 352(LMCT), 296,232,(π-π*) nm. IR (KBr Disc): 3300 (vb), 3095(s), 2950(s), 1712(w),1574(w), 1425(s), 1431(m), 1373(m) 1363(w), 1182(m), 1128(w), 1024(m),1014(m), 887(m), 825(vs) cm⁻¹.

VCp₂X₂. The pseudo-halide derivatives with X=N₃ ⁻ (Compound 8), CN⁻(Compound 9), OCN⁻ (Compound 10), and SCN⁻ (Compound 12) were preparedby following the procedure described in Doyle, G., Tobias, R. S.Pseudohalide and chelate complexes of bis(cyclopentadienyl)vanadium(IV).Inorg. Chem., 7: 2479-2484, 1968. The pure compounds were isolatedeither recrystallization or from Soxhlet extraction. The purity of thesecomplexes were checked by elemental analysis, melting point data andUV-visible and IR spectrum. The results are given below:

VCp₂(N₃)₂, (Compound 8). Yield: 65%. M.P. Sublimes at 173° C.(decomposes). Anal. Calcd. for VC₁₀H₁₀N₆: C, 45.28, H, 3.77; N, 31.2.Found: C, 45.28; H, 3.73; N, 31.16. UV-vis: (CH₂Cl₂) λ_(max): 434 (d—d),314 (LMCT), 257, 233 (π-π*) nm. IR (KBr Disc): 3125(m), 3111(m),3104(m), 3079(m), 2067(vs), 2031(vs), 1448(m), 1375(m), 1330(m),1280(m), 1126(w), 1080(w), 1024(m), 835(vs), 646(w), 590(m), 438(m),400(w) cm⁻.

VCp₂(CN)₂, (Compound 9). Yield: 75%. M.P. Sublimes at 173° C.(decomposes). Anal. Calcd. for VC₁₂H₁₀N₂: C, 61.89, H, 4.29; N, 12.0.Found: C, 60.98; H, 4.30; N, 11.45. UV-vis: (CH₂Cl₂) λ_(max): 605 (d—d),394 (LMCT), 307, 250 (π-π*) nm. IR (KBr Disc): 3450(m,b), 3114(vs),2120(s), 2110(s), 1435(s), 1420(s), 1369(m), 1373(w), 1126(m), 1014(s),881(s), 858(vs), 845(vs), 480(m), 400(m) cm⁻¹.

VCp₂(NCO)₂, (Compound 10). Yield: 55%. M.P. 287° C. (decomposes). Anal.Calcd. for VC₁₂H₁₀N₂O₂: C, 54.3, H, 3.8; N, 10.6. Found: C, 53.85; H,3.97; N, 10.2. UV-vis: (CH₂Cl₂) λ_(max): 742 (d—d), 373 (LMCT), 277, 237(π-π*) nm. IR (KBr Disc): 3531(m), 3110(m), 2657(w), 2248(vs), 2117(vs),2170(vs), 1330(s), 1024(w), 833(vs), 603(s), 593(s), 420(m) cm⁻¹.

VCp₂(NCO)Cl, (Compound 11). The dark brown powder was isolated byfollowing the procedure described for the Titanium analogue. Köpf-Maier,P., Grabowski, S., Köpf, H. Tumorhemmung durch Metallocene:Titan-Komplexe des type [TiCp₂XY] und [TiCpX₂Y]. Eur. J.Med.Chem-Chim.Ther. 19: 347-352, 1984. Anal. Calcd. for VC₁₁H₁₀NOCl: C, 51.06, H,3.87; N, 5.41, Cl, 13.73. Found: C, 51.35; H, 3.97; N, 5.65, Cl, 13.45.UV-vis: (CH₂Cl₂) λ_(max): 710 (d—d), 490 (LMCT), 257, 227 (π-π*) nm. IR(KBr Disc): 3513(sp,w), 3110(m), 2657(w), 2117(vs), 1444(m), 1330(s),1261(w), 1018(m),950(m), 833(vs), 635(s), 424(w) cm⁻¹.

VCp₂(NCS)₂. 0.5H₂O, (Compound 12). Yield: 75%. M.P. The compoundsublimes at 150° C. (decomposes). Anal. Calcd. for VC₁₂H₁₁N₂O_(1/2)S₂:C,47.05, H, 3.59; N, 9.15, S, 20.91. Found: C, 47.55; H, 3.26; N, 9.06; S,20.91. UV-vis: (CH₂Cl₂) λ_(max): 739 (d—d), 401 (LMCT), 463 (SCN⁻:π-π*), 251, 270 (π-π*) nm. IR (KBr Disc): 3400(w,b), 3087(s), 2086(vs),2067(vs), 1433(s), 1423(m), 1010(m), 1070(m) 840(vs), 480(vw), 410(m)cm⁻¹.

VCp₂(NCSe)₂, (Compound 13). To a stirring solution of VCp₂Cl₂, (0.4 g,1.6 mmol) in anhydrous acetone (25 ml) under argon, was added solidKNCSe (0.85 g, 8 mmol). The reaction mixture was allowed to stir for 4 hat room temperature. The resulting red brown solution was subjected torotatory vaporization and the pure microcrystalline red compound wasisolated from the crude product through Soxhlet extraction usingdichloromethane as solvent. Yield: 60%. M.P. The compound slowly turnsblack, decomposes at 250-275° C. (decomposes). Anal. Calcd. forVC₁₂H₁₀N₂Se₂:C, 36.83, H, 2.56; N, 7.1. Found: C, 36.85; H, 2.64; N,6.97. UV-vis: (CH₂Cl₂) λ_(max): 716 (d—d), 456 (LMCT), 488 (SeCN⁻:π-π*), 251, 270 (π-π*) nm. IR (KBr Disc): 3475(m), 3076(s), 2085(vs),2065(vs), 1444(m), 1431(s), 1126(w), 1074(w), 1008 (m), 962 (m) 843(vs)cm⁻¹.

VCp₂Cl (CH₃CN)(FeCl₄), (Compound 14). Compound (14) was prepared byfollowing the procedure described for the corresponding titaniumcomplex. Neuse, E. W., Meirim, M. G. A chlorotitanocenetetrachlorferrate complex stabilized by acetonitrile coordination.Transition Met. Chem., 9: 337-338, 1984. In the instant synthesis, the1:1.1 stoichiometric mole ratio between VCp₂Cl₂ and anhydrous FeCl₃solution was strictly maintained in acetonitrile solution. The darkgreen precipitate was isolated from the reduced volume of the parentsolution after overnight standing at −20° C. Yield: 90%. M.P. Notdetermined. Anal. Calcd. for: VC₁₂H₁₃NCl₅Fe: C, 31.6; H, 2.9; N, 3.1;Cl, 39.98. Found: yet to receive the data. UV-Vis : (CH₃CN) λ_(max):648, 575, (d—d), 362 (Superimposed bands of LMCT of Cp₂V²⁺ and FeCl₄ ⁻),311(FeCl₄ ⁻, LMCT), 265(sh), (π-π* of Cp rings), 240(superimposed bandsof π-π* of Cp rings and FeCl₄ ⁻ nm. IR (KBr Disc): 3386(sb), 3109(m),2924(m), 2360(w), 2318(s), 2289(m), 1622(m), 1447(s), 1435(m), 1358(w),1128(m), 1027(s), 1012(s), 856(vs), 846(s) cm⁻.

VCp₂(O₃SCF₃)₂, (Compound 15). The generation of VCp₂(O₃SCF₃)₂ in THFsolution was induced by following the procedure that was described forTitanium analogue. Thewalt, U., Berhalter, K. Kationische Komplexe MitDer (η⁵-C₅H₅)₂Ti^(IV)-Baugruppe: Darstellung und Struktur Von[(η⁵-C₅H₅)₂ Ti(bpy)]²⁺ (CF₃SO₃ ⁻)₂. J. Organometallic Chem., 302:193-200, 1986. The precipitated silver chloride was removed byfiltration and the filtrate evaporated to dryness. The solid greenresidue was redissolved in 20 mL of CH₂Cl₂, filtered again throughcannula with one end covered with filter paper—cotton assembly, securelytightened by fine bore copper wire. The dark green precipitate wasisolated from dichloromethane using diethyl ether as a cosolvent. Thecompound is moisture-sensitive. Yield: 40%, m.p. at 137° C.decomposition starts. Anal. Calcd. for VC₁₂H₁₀S₂O₆F₆: C, 30.06; H, 2.09;S, 13.36. Found: C, 29.98; H, 2.18; S, 13.19. UV-Vis: (DMSO) λ_(max):620 (d—d), 379 (LMCT), 286, 261(π-π*) IR (KBr Disc): 3400(s,b), 3093(m),1635(m), 1446(s), 1436(s), 1259(vb,d) 1178(vs), 1033(vs), 885(s),822(vs), 770(m), 643(vs,d), 580, (m), 518(vs) cm⁻¹.

VCp₂(acac)(O₃SCF₃), (Compound 16). Dark black colored large crystalswere obtained by following the literature procedure. Doyle, G., Tobias,R. S. Pseudohalide and chelate complexes ofbis(cyclopentadienyl)vanadium(IV). Inorg. Chem., 7: 2479-2484, 1968.Yield: 45%, M.P. 247° C. decomposition starts. Anal. Calcd. forVC₁₆H₁₇SO₅F₃: C, 44.45; H, 3.96; S, 7.46. Found: C, 44.81; H, 3.99; S,7.52. UV-Vis : (CH₂Cl₂) λ_(max): 740, 640 (d—d), 370 (LMCT), 309 (π-π*of acac⁻ moiety), 270, 230(π-π* of Cp⁻ rings). IR (KBr Disc): 3118(s),2295(w), 1564(vs), 1516(vs), 1440(s), 1350(s), 1267(s,b) 1194(w),1149(vs) 1067(w), 1032(s), 983(w), 959(w), 910(w), 843(vs), 783(vs),756(m), 638(m), 573(vs), 456(s), 408 (m) cm⁻¹.

VCp₂(Hfacac)(O₃SCF₃), (Compound 17). Micro dark green powder wasisolated following the procedure described Doyle, G., Tobias, R. S.Pseudohalide and chelate complexes of bis(cyclopentadienyl)vanadium(IV).Inorg. Chem., 7: 2479-2484, 1968. Yield: 20%, M.P. 225° C. decompositionstarts. Anal. Calcd. for VC₁₆H₁₁SO₅F₉: C,35.89; H, 2.06; S, 5.98. Found:C, 35.76; H, 2.08; S, 5.89. UV-Vis: (CH₂Cl₂) λ_(max): 700, 557 (d—d),377 (LMCT), 314 (π-π* of Hfacac⁻ moiety), 271, 244(π-π* of Cp⁻ rings).IR (KBr Disc): 3117(m), 1637(vs), 1597(w), 1552(w), 1523(w), 1446 (s),1358(w), 1267(s,b) 1194(w), 1149(vs) 1067(w), 1032(s), 983(w), 959(w),910(w), 843(vs), 783(vs),756(m), 638(m), 573(vs), 456(s), 408(m) cm⁻¹.

VCp₂(bpy)(O₃SCF₃)₂, (Compound 18). The synthetic procedure was amodified procedure described for TiCp₂(bpy)(O₃SCF₃)₂. Thewalt, U.,Berhalter, K. Kationische Komplexe Mit Der (η⁵-C₅H₅)₂ Ti^(IV)-Baugruppe:Darstellung und Struktur Von [(η⁵-C₅H₅)₂ Ti(bpy)]²⁺ (CF₃SO₃)₂. J.Organometallic Chem., 302: 193-200, 1986. Light grayish powder wasobtained as a precipitate from the THF solution which was collected byfiltration and dried. Yield: 38%. M.P.305° C. Anal Calcd. forVC₂₄H₂₀N₂F₆O₆VS: C, 53.1; H, 3.69N, 2.58; S, 5.9. Found: C, 52.48; H,3.72; N, 2.51; S, 5.73. UV-Vis (DMSO) λ_(max): 780(sh), 555(d—d),326(LMCT of Cp₂V²⁺), 272 (sh) (π-π* of Cp ring), 241 (superimposed bandsof π-π* of Cp rings and bipyridine) nm. IR (cm−1): 3135(m), 3099(s),1605(vs), 1504(m), 1477(s), 1452(s), 1437(vs), 1307(m), 1257(vs)1232(vs), 1207(m), 1028(vs), 862(vs), 837(w), 771(vs), 636(vs), 517(vs),435(vw) cm⁻¹.

Cp₂V(cat), (Compound 19). Cp₂VCl₂ (126 mg, 0.50 mmol) was placed in a250 mL flask and dissolved in THF (100 mL). In another flask the sodiumcatecholate (Cat) was prepared by the addition of NaH (25 mg, 1.0 mmol,mineral oil had been previously removed by washing with petrolium ether)catechol (to 55.5 mg, 0.50 mmol) in THF (15 mL). The solution wasstirred for 2 hours, resulting in a deep blue solution. The catecholatesolution was cannulated into the vanadium solution and stirred for 4hours. The reaction mixture was opened to the air and quickly flashchromatographed under nitrogen on alumina (neutral) (acetonitrile mobliephase). The solvent of the deep blue solution was then removed undervacuum and the product collected. Yield: 26%. M.P. Not determined. Anal.Calcd. for VC₁₆H₁₄O₂: C, 66.45; H, 4.88. Found: C, 66.79; H, 4.93.UV-vis (CH₃CN) λ_(max): 711 (2078), 438 (2041) (LMCT of Cat—V(IV)), 337(3173) (LMCT of Cp—V(IV)), 292 (8564), 275 (14661), 259 (18945) (π-π* ofCat and Cp rings). IR (KBr pellet): 3100 (w), 3080 (w), 2951(m),2945(w), 2860(w), 1468(s), 1438(m), 1404(m), 1359(w), 1261(vs), 1012(w),929(w), 804(vs), 638(w) cm⁻¹.

VCp₂(dtc), (Compound 20). Bis(cyclopendienyle)N, N-diethyldithiocarbamato triflate salt was prepared according to the publishedprocedure. Casey, A. T., Thackeray, J. R. Dithiochelates of theBis(h-cyclopentadienyl)vanadium (IV) moiety. IIN,N-Dialkyldithiocarbamate and O,O′-dialkyldithiophospahte complexes.Aust. J. Chem. 27: 757-768, 1974. Yield: 90%. M.P. 163° C. Anal. Calcd.for VC₁₆H₂₀S₃F: C, 48.09; H, 3.93; N, 2.75; S, 24.36. Found: C, 48.11;H, 3.97; N, 2.79; S, 24.41. UV-vis (CH₃CN) λ_(max): 621, 535 (d—d), 392(LMCT), 330 (Dtc⁻: π-π*), 276, 270, 230 (π-π* of Cp and Dtc⁻). IR (KBrpellet): 3107(m), 1632(s), 1595(w), 1538(w), 1439(s), 1212(s), 1201(s),1156(s), 1123(m), 1019(s), 855(s), 641(s) cm⁻¹.

VCp₂(PH), (Compound 21). The reaction mixture composed of VCp₂Cl₂ (0.2g, 8 mmol) and AgCF₃SO₃ (0.46 g, 18 mmol) in H₂O (10 mL) was stirred for2 h and then filtered through fine glass frit. A solution of N-phenylbenzohydroxamic acid in 5 mL ethanol, 0.85 g, 4.0 mmol, was added to thefiltrate with stirring, and the resulting solution was kept for 4 h tocomplete the precipitation of dark colored compound. These werecollected by filtration and thoroughly washed with diethyl ether anddried for overnight under vacuum. Yield: 38%. M.P. 160° C. Anal Calcd.for VC₂₄H₂₀NF₃O₅S: C, 53.1; H, 3.69; N, 2.58; S, 5.9. Found: C, 52.48;H, 3.72; N, 2.51; S, 5.73. UV-Vis: (CH₂Cl₂) λ_(max): 680, 501 (d—d), 377(LMCT), 314 (π-π* of hydroxamate moiety), 261, 233 (π-π* of Cp ring) nm.IR (cm⁻¹): 3345(sb), 3117(s), 1651(mb), 1600(m), 1539((vs), 1495(m),1450(m), 1300(m), 1281(s), 1244(vs), 1173(s), 999(m), 758(m), 694((m),638((s), 578(w), 515(m) cm⁻¹.

VCp₂(H), (Compound 22). This reddish-brown compound was preparedfollowing the procedure applied for the compound (20). D'Cruz, O. J.,Ghosh, P., Uckun, F. M. Antitumor activity of chelated complexes ofbis(cyclopentadienyl)vanadium(IV). Mol. Hum. Reprod., 4: 683-693, 1998.In the instant synthesis, the reactions were carried out in dry THFinstead of H₂O using acethydroxamic acid as ligand. Yield: 52%. M.P.Could not be determined because it absorbs moisture from the air andturns pasty within few minutes. Anal Calcd. for VC₁₃H₁₄NF₃O₅S: C, 38.61;H, 3.46; N, 3.46; S, 7.92. Found: C, 38.12; H, 3.72; N, 3.26; S, 7.81.UV-Vis: (CH₂Cl₂) λ_(max): 710, 550 (d—d), 401 (LMCT), 300 (π-π* ofhydroxamate moiety), 261, 233 (π-π* of Cp ring)nm. IR (Kbr Disc):3345(s,vb), 1695(mb), 1635(m), 1500((vs), 1450(s), 1280(m), 1260(s),1215 (vs), 1144(s), 959(m), 758(m), 635((m), 540(w), 480(m) cm⁻.

V(MeCp)₂Cl₂. 0.5 H₂O, (Compound 23). The synthetic procedure isdescribed in the literature. Petersen, J. L., Dahl, L. F. Synthesis andstructural characterization by X-ray diffraction and electronparamagnetic resonance single-crystal techniques of V(η⁵-C₅H₄CH₃)₂Cl₂. Astudy of the spatial distribution of the unpaired electron in aV(η⁵C₅H₅)₂L₂-type complex. J. Am. Chem. Soc., 97: 6422-6433, 1975. Thebright green microcrystals were seperated from the HCl saturated CHCl₃solution. Yield. 25%. Anal Calcd. for VC₁₂H₁₅Cl₂O_(0.5): C, 49.82; H,5.19; Cl, 24.60. Found: C, 49.92; H, 34.90; Cl, 24.90. UV-Vis (CH₂Cl₂)λ_(max): 760, 659 (d—d), 383 (LMCT of Cp₂V²⁺), 286, 233 (π-π* of MeCprings), nm. IR (cm−1): 3135(m), 3099(s), 1307(m), 1028(vs), 862(vs),771(vs), 636(vs), 517(vs) cm⁻¹.

V(Me₅Cp)₂Cl₂, (Compound 24). The green solid was isolated fromdiethylether from a reaction mixture of V(Me₅Cp)₂ and PCl₃ as describedby Moran, M., Masaguer, J. R., Fernandez, V. Synthesis andcharacterization of halogen and pseudohalogen derivatives of substitutedvanadocenes. J.Organometallic Chem. 291: 311-319, 1985. Yield. 20%. AnalCalcd. for VC₂₀H₃₀Cl₂: C, 61.2; H, 7.5; Cl, 13.0. Found: C, 59.9; H,7.5; Cl, 13.1. UV-Vis (CH₂Cl₂) λ_(max): 740, 652 (d—d), 440 (LMCT ofCp₂V²⁺), 270, 230 (π-π* of Me₅Cp-rings), nm. IR (Kbr Disc): 3118(s),1194(w), 1149(m), 1032(s), 959(w), 843(vs), 638(vs) cm⁻¹.

V(Me₅Cp)OCl, (Compound 25). This compound was prepared by following theprocedure reported in Aistars, A., Newton, C., Rübensthal, T., Doherty,N. M. Covenient synthesis ofdichloro(oxo)(pentamethylcyclopentadienyl)vanadium(V), (ζ-C₅Me₅)V(O)Cl₂.Organometallics. 16: 1994-1996, 1997. Sublimed green materials ofV(Me₅Cp)₂Cl₂ (24) was dissolved in dry THF and were subjected to purgedwith O₂ for 8 h. The solvent was removed under vacuum and recrystallizedfrom hexane. Yield. 80%. Anal Calcd. for VC₁₀H₁₅OCl₂: C, 43.98; H, 5.54;Cl, 27.67. Found: C, 43.29; H, 5.62; Cl, 27.54. ¹HNMR, ⁵¹V NMR: δ 2.335(5×CH₃). (CH₂Cl₂) (UV-Vis (CH₂Cl₂) λ_(max): 680, 605(LMCT of Oxygen andchloride ligand) 410 (LMCT of Cp to V(O)²⁺), 290, 250 (π-π* ofMe₅Cp-ring), nm. IR (Kbr Disc): 3440(b, m), 2962(m), 2906(s), 2858(m),1621(w), 1487(m), 1437(s), 1375(vs), 1066(s), 1014(m), 960(m), 943(m),806(m), 744(m), 700(m) cm⁻¹.

In vitro Invasion Assays

The in vitro invasiveness of vanadocene-treated cancer cells was assayedusing a previously published method which employs Matrigel-coated Costar24-well transwell cell culture chambers (“Boyden chambers”) with8.0-μm-pore polycarbonate filter inserts. Narla R K, Liu X P, Klis D,Uckun F M. Inhibition of human glioblastoma cell adhesion and invasionby 4-(4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131) and4-(3′-bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline(WHI-P154). Clin Cancer Res 4:2463-71, 1998. Albini, A., Iwamoto, Y.,Kleinman, H. K., Martin, G. R., Aaronson, S. A., Kozlowski, J. M., andMcEwan, R. N. A rapid in vitro assay for quantitating the invasivenessof tumor cells. Cancer Res. 47: 3239-3245, 1987. The chamber filterswere coated with 50 μg/ml of Matrigel matrix, incubated overnight atroom temperature under a laminar flow hood and stored at 4° C. On theday of the experiment, the coated inserts were rehydrated with 0.5 mlserum-free DMEM containing 0.1% bovine serum albumin for 1-2 hours. Tostudy the effects of vanadocenes VDC and VDSe on invasiveness of cancercells, exponentially growing cells were incubated with these vanadocenesat various concentrations ranging from 1 μM to 10 μM overnight. Thecells were trypsinized, washed twice with serum-free DMEM containingBSA, counted and resuspended at 1×10⁵ cells/ml. 0.5 ml cell suspensioncontaining 5×10⁴ cells in a serum-free DMEM containing vanadocenecompounds or vehicle was added to the Matrigel-coated and rehydratedfilter inserts. Next, 750 μl of NIH fibroblast conditioned medium wasplaced as a chemoattractant in 24-well plates and the inserts wereplaced in wells and incubated at 37° C. for 48 hr. After the incubationperiod, the filter inserts were removed, the medium was decanted off andthe cells on the top side of the filter that did not migrate werescrapped off with a cotton tipped applicator. The invasive cells thatmigrated to the lower side of the filter were fixed, stained with Hema-3solutions and counted under microscope. Five to 10 random fields perfilter were counted to determine the mean (±SE) values for the invasivefraction. The invasive fractions of cells treated with quinazolinederivatives were compared to those of DMSO treated control cells and thepercent inhibition of invasiveness was determined using the formula: %Inhibition=100×(1−Invasive Fraction of Drug-Treated Cells/InvasiveFraction of Control Cells). Each treatment condition was evaluated induplicate in 3 independent experiments. IC50 values were calculated bynon-linear regression analysis using an Graphpad Prism software version2.0 (Graphpad Software, Inc., San Diego, Calif.).

Apoptosis Assays

A flow cytometric two-color terminal dideoxynucleotidyl transferase(TdT)-mediated digoxigenin-uridine triphosphate (dUTP) nick-end labelingassay (TUNEL) was employed to detect apoptotic nuclei. Gavrieli, Y.,Sherman, Y., Ben-Sasson, S. A. Identification of programmed cell deathin situ via specific labeling of nuclear DNA fragmentation. J. CellBiol., 119: 493-501, 1992. Exponentially growing cells (10⁶/ml) wereincubated in DMSO alone (0.1%) or treated with 100 μM each of the 16vanadocenes (VDB, VDC, VMDC, VDI, VDA, VDCN, VDOCN, VDSCN, VDSeCN, VDT,VDCO, VDFe, VD(acac), VDH, VD(dpy), and VD(dtc) in 0.1% DMSO for 24 h.Cells were washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 minon ice. Following two washings in PBS, they were permeabilized with 0.1%Triton X-100 in 0.1% sodium citrate for 2 min on ice, and washed twicein PBS. Labeling of exposed 3′-hydroxyl (3′-OH) ends of fragmentednuclear DNA was performed using TdT and fluorescein isothiocyanate(FITC)-conjugated dUTP according to the manufacturer's recommendations(Boehringer Mannheim, Indianapolis, Ind.). Cells were counterstainedwith 5 μg/ml PI. Control samples included: (i) untreated cells; (ii)cells incubated with the reaction mixture without the TdT enzyme. Cellswere analyzed with a FACS Calibur flow cytometer (Becton Dickinson,Mountain View, Calif.). Relative DNA content (PI) was detected withband-pass filter 585/42, and dUTP incorporation (FITC) was detected withband-pass filter 530/30. Fluorescence was compensated for in theacquisition software using single-label control samples. Data wereacquired in listmode, gated to 10,000 events per sample, and analyzedwith the use of CELLQuest software program (Becton Dickinson).Nonapoptotic cells do not incorporate significant amounts of dUTP due tolack of exposed 3′-OH ends, and consequently have relatively little orno fluorescence compared to apoptotic cells which have an abundance of3′-OH (M2 gates). Vanadocene-induced apoptosis is shown by an increasein the number of cells staining with FITC-dUTP. The M1 and M2 gates wereused to demarcate non-apoptotic and apoptotic PI-counterstained cellpopulations, respectively. TUNEL assays were performed using twotesticular cell lines Tera-2 and Ntera-2 following exposure to each ofthe 16 vanadocenes. In other experiments, MC540 binding (as an earlymarker of apoptosis) and PI permeability (as a marker of advanced stageapoptosis) were simultaneously measured in human cancer cells 24 hoursafter exposure to vanadocenes, as previously described. Uckun, F. M.,Narla, R. K., Jun, X., Zeren, T., Venkatachalam, T., Waddick, K. G.,Rostostev, A., Myers, D. E. Cytotoxic activity of EGF-genstein againstbreast cancer cells. Clin. Cancer Res. 4:901-912, 1998. Vassilev, A.,Ozer, Z., Navara, C., Mahajan, S., and Uckun, F. M. Bruton's tyrosinekinase as an inhibitor of the Fas/CD95 death-inducing signaling complex.J. Biol. Chem. 274:1646-56, 1999. Whole cells were analyzed using aFACStar Plus flow cytometer (Becton Dickinson, San Jose, Calif.). Allanalyses were done using 488 nm excitation from an argon laser. MC540and PI emissions were split with a 600 nm short pass dichroic mirror anda 575 nm band pass filter was placed in front of one photomultipliertube to measure MC540 emission and a 635 nm band pass filter was usedfor PI emission.

Morphological evidence for apoptosis was sought among the TUNEL-positivecells using confocal laser scanning microscopy (CLSM). Confocalmicroscopy was performed using BioRad MRC-1024 Laser Scanning ConfocalMicroscope (BioRad, Hercules, Calif.) equipped with a krypton/argonmixed gas laser (excitation lines at 488, 568, and 647 nm) and mountedon a Nikon Eclipse E800 series upright microscope equipped with highnumerical objectives. Using fluorescence imaging, the fluorescenceemission of FITC and PI from nuclei of in Ntera-2 cells wassimultaneously recorded using 598/40 nm, and 680 DF32 emission filterrespectively. Confocal images were obtained using a Nikon 60×(NA 1.4)objective and Kalman collection filter. Digitized images were saved on aJaz disk (lomega Corp., Roy, Utah) and processed with the AdobePhotoshop software (Adobe Systems, Mountain View, Calif.). Final imageswere printed using a Fuji Pictography 3000 (Fuji Photo Film Co., Tokyo,Japan) color printer.

In other experiments, TERA-2 and NTREA-2 cells were plated at 50%confluency in T-150 flasks in media supplemented with 10% FCS; 24 hlater, they were exposed to vehicle (0.05% DMSO) or 100 μM each of the 6representative vanadocenes (VDC, VDO, VDSCN, VDSeCN, VDT, and VD(dtc) in0.05% DMSO. Combined adherent and nonadherent cells (5×10⁶/sample) wereharvested at 24 h and washed in PBS. DNA was prepared from Triton-X-100lysates for analysis of fragmentation. Uckun, F. M., Evans, W. E.,Forsyth, C. J., Waddick, K. G., Tuel-Ahlgren, L., Chelstrom, L. M.,Burkhardt, A., Bolen, J., Myers, D. E. Biotherapy of B-cell precursorleukemia by targeting genistein to CD19-associated tyrosine kinase.Science (Washington D.C.) 267:886-91, 1995. Uckun, F. M., Narla, R. K.,Jun, X., Zeren, T., Venkatachalam, T., Waddick, K. G., Rostostev, A.,Myers, D. E. Cytotoxic activity of EGF-genstein against breast cancercells. Clin. Cancer Res. 4:901-912, 1998. In brief, cells were lysed inhypotonic 10 mmol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA, 0.2% Triton-X-100detergent; and subsequently centrifuged at 11,000 g. To detectapoptosis-associated DNA fragmentation, supernatants wereelectrophoresced on a 1.2% agarose gel, and the DNA fragments werevisualized by ultraviolet light after staining with ethidium bromide. Inadditional experiments, (a) leukemia cell lines NALM-6, MOLT-3, andHL-60, (b) glioblastoma cell lines U373 and U87, and (c) breast cancercell lines MDA-MB-231 and BT-20 were exposed to multiple concentrationsof VDC and VDSeCN and then assayed for apoptosis by DNA gels as well asflow cytometry.

In some experiments, immunofluorescence was used to examine themorphologic features of vanadocene-treated cancer cells. At the end ofthe indicated treatment period, cells were washed twice with PBS andfixed in 2% paraformaldehyde. The cells were permeabilized andnon-specific binding sites were blocked with 2.5% BSA in PBS containing0.1% Triton X-100 for 30 min. Tubulin expression was examined byimmunofluorescence using a monoclonal antibody against α-tubulin (SigmaChemical Co, St. Louis, Mo.) at a dilution of 1:1000 and an anti-mouseIgG conjugated to FITC. Cells were washed in PBS and counterstained withtoto-3 (Molecular Probes Inc., Eugene, Oreg.) for 10 min at a dilutionof 1:1000. Cells were washed again with PBS and the coverslips weremounted with Vectashield (Vector Labs, Burlingame, Calif.) and viewedwith a confocal microscope (Bio-Rad MRC1024) mounted in a NikonLabhophot upright microscope. Digital images were saved on a Jaz diskand processed with Adobe Photoshop software (Adobe Systems, MountainView, Calif.).

Oxovanadium

Cytotoxicity Assays

The cytotoxicity of oxovanadium (IV) complexes listed in Table 6 weretested against 9 different human cancer cell lines was performed usingthe MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)assay (Boehringer Mannheim Corp., Indianapolis, Ind.) as describedpreviously (20). Briefly, exponentially growing tumor cells were seededinto a 96-well plate at a density of 4×10⁴ cells/well and incubated withmedium containing the oxovanadium(IV) compounds concentrations rangingfrom 0.1 to 250 μM for 48 hours at 37° C. in a humidified 5% CO₂atmosphere. Triplicate wells were used for each treatment. To each well,10 μl of MTT (0.5 mg/ml final concentration) was added and the plateswere incubated at 37° C. for 4 hours to allow MTT to form formazancrystals by reacting with metabolically active cells. The formazancrystals were solubilized overnight at 37° C. in a solution containing10% SDS in 0.01 M HCl. The absorbence of each well was measured in amicroplate reader (Labsystems) at 540 nm and a reference wavelength of690 nm. To translate the OD₅₄₀ values into the number of live cells ineach well, the OD₅₄₀ values were compared to those on standardOD₅₄₀—versus—cell number curves generated for each cell line. Thepercent survival was calculated using the formula: % survival=Live cellnumber[test]/Live cell number [control]×100. The IC₅₀ values werecalculated by non-linear regression analysis using an Graphpad Prismsoftware version 2.0 (Graphpad Software, Inc., San Diego, Calif.).

In situ Detection of Apoptosis

The demonstration of apoptosis was performed as described earlier (20,21) by the in situ nick-end-labeling method using in situ cell deathdetection kit (Boehringer Mannheim Corp., Indianapolis, Ind.) accordingto the manufacturer's recommendations. Exponentially growing cells wereseeded in 6-well tissue culture plates and incubated with fresh mediumcontaining compounds. After a 24 hour incubation at 37° C. in ahumidified 5% CO₂ incubator the cells were collected into a 15 mlcentrifuge tube, washed with PBS and pelleted by centrifugation at 1000rpm for 5 min. The cells were fixed in 2% paraformaldehyde, washed withPBS and pelleted by centrifuging the tubes at 1000 rpm for 5 min. Cellspellets were resuspended in 50 μl of PBS, transferred to superforst plusslides and allowed to attach for 15 min. The cells were permeabilizedwith 0.1% triton X-100 in 0.1% citrate buffer and incubated for 1 hr at37° C. with the reaction mixture containing terminal deoxynucleotidyltransferase (TdT) and fluorescein isothiocyanate (FITC)-conjugated dUTP.Cells were washed with PBS to remove unbound reagents and the coverslipswere mounted onto slides with Vectashield containing propidium iodide(Vector Labs, Burlingame, Calif.) and slides were viewed with a confocallaser scanning microscope. Non-apoptotic cells do not incorporatesignificant amounts of dUTP due to lack of exposed 3-hydroxyl ends, andconsequently have much less fluorescence than apoptotic cells which havean abundance of exposed 3′-hydroxyl ends. In control reactions, the TdTenzyme was omitted from the reaction mixture.

Mitochondrial Transmembrane Potential Assessment

To measure the changes in mitochondria, NALM-6 cells were incubated withcompound 29 at concentrations ranging from 0.1 μM to 1 μM for 24 hr, 48hr, 72 hr or 96 hr. stained with specific fluorescent dyes and analyzedwith flow cytometer. Mitochondrial membrane potential (ΔΨm) was measuredusing two dyes including a lipophollic cation5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethlybenzimidazolylcarbocyanineiodide (JC-1; Molecular probes, Eugene, Oreg.) and a cyanine dye,1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide [DiIC₁(5); Molecularprobes] as earlier described (22). JC-1 can enter the cells, andselectively mitochondria, and has been used to assess ΔΨm in a varietyof studies (23,24). JC-1 is a monomer at 527 nm after being excited at490 nm; with polarization of ΔΨm, J-aggregates are formed that shiftemission to 590 nm (23, 24). This can be detected on a flow cytometer byassessing the green signal (at 527 nm) and green-orange signal (at 590nm) simultaneously, creating an index of the number of cells polarizedand depolarized mitochondria. The treated and control cells were washedand plated in a 6-well plate at 1×10⁶ cells in fresh medium. JC-1 wasadded at a concentration of 10 μg/ml, and cells were incubated for 10min in the dark at room temp. Cells were then washed twice with ice-coldPBS and immediately analyzed using Becton Dickinson (San Jose, Calif.)Calibur flow cytometer. At least 2×10³ cells were analyzed to determinethe percentage of cells with polarized and depolarized mitochondria.DiIC₁(5), a cyanine dye is amphioatheic and cationic that concentrate inenergized mitochondria and has been used in a variety of studies tomeasure the mitochondrial membrane potential (25-27). NALM-6 cells werestained with DiIC₁ (5) at 40 nM concentration for 30 min in the dark asdescribed for JC-1. The cells were analyzed using Vantage BectonDickinson cell sorter equipped with HeNe laser with excitation at 635 nmand the fluorescence was collected at 666 nm.

Mitochondrial Mass Determination

Relative mitochondrial mass was measured by using Becton DickinsonCalibur flow cytometry and the fluorescent stain 10-n-nonyl-acridineorange (NAO), which binds the mitochondrial phospholipid cardiolipin,that has been extensively used to provide an index of mitochondrial mass(28). 1×10⁶ compound 29-treated NALM-6 cells at concentrations of 0.1 μMto 1 μM for 24 hr, 48 hr, 72 hr, or 96 hr were incubated with 30 μM ofNAO in complete medium for 10 min at room temp in the dark, washed withice-cold PBS and analyzed using log scale photomultiplier to detect thegreen fluorescence at 527 nm. Relative change in the mitochondrial masswas measured using the mean fluorescence value of mitochondria ofcompound 29-treated and vehicle-treated cells.

Quantitative Apoptosis Assays

A flow cytometric two-color terminal dideoxynucleotidyl transferase(TdT) mediated digoxigenin-uridine triphosphate (dUTP) nick-end labelingassay (TUNEL) was employed to detect apoptotic nuclei. Edelman, G. M.Adhesion and counteradhesion: morphogenetic functions of the cellsurface. Prog. Brain Res. 101: 1-14, 1994. Exponentially growing cells(10⁶/ml) were incubated in DMSO alone (0.1%) or treated with 50 μM eachof the 15 oxovanadium compounds in 0.1% DMSO for 24 h. Cells were washedin PBS, fixed in 4% paraformaldehyde in PBS for 15 min on ice. Followingtwo washings in PBS, they were permeabilized with 0.1% Triton X-100 in0.1% sodium citrate for 2 min on ice, and washed twice in PBS. Labelingof exposed 3′-hydroxyl (3′-OH) ends of fragmented nuclear DNA wasperformed using TdT and fluorescein isothiocyanate (FITC)-conjugateddUTP according to the manufacturer's recommendations (BoehringerMannheim, Indianapolis, Ind.). Cells were counterstained with 5 μg/mlPI. Control samples included: (i) untreated cells; (ii) cells incubatedwith the reaction mixture without the TdT enzyme. Cells were analyzedwith a FACS Calibur flow cytometer (Becton Dickinson, Mountain View,Calif.). Relative DNA content (PI) was detected with band-pass filter585/42, and dUTP incorporation (FITC) was detected with band-pass filter530/30. Fluorescence was compensated for in the acquisition softwareusing single-label control samples. Data were acquired in listmode,gated to 10,000 events per sample, and analyzed with the use ofCELLQuest software program (Becton Dickinson). Nonapoptotic cells do notincorporate significant amounts of dUTP due to lack of exposed 3′-OHends, and consequently have relatively little or no fluorescencecompared to apoptotic cells which have an abundance of 3′-OH (M2 gates).Oxovanadium compounds-induced apoptosis is shown by an increase in thenumber of cells staining with FITC-dUTP. The M1 and M2 gates were usedto demarcate non-apoptotic and apoptotic PI-counterstained cellpopulations, respectively.

RESULTS Vanadocene Compounds

Synthesis and Characterization of Vanadocene Compounds.

The chemical structures and physical data (viz., UV-vis spectral data,infrared spectral data, and elemental analysis results) of compounds1-25 are detailed in Table 1, Table 2, Table 3, and Table 4.

Cytotoxic Effects of Vanadocenes on Human Testicular Cancer Cells

Using the mitochondrial function-based MTT viability assay, the effectsof 5 metallocene dichlorides containing vanadium (VDC), titanium (TDC),zirconium (ZDC), molybdenum (MDC), or hafnium (HDC), on two humantesticular cancer cell lines, Tera-2 and Ntera-2, were tested bymeasuring cellular proliferation at 7 different concentrations rangingfrom 1.9 μM to 250 μM for 24 h (FIG. 1). Only vanadium-containingmetallocene, VDC, inhibited the growth of both cell lines with IC₅₀values of 80.6 μM and 74.0 μM, respectively. Surprisingly, othermetallocene dichlorides containing titanium, zirconium, molybdenum orhafnium as central metal atom (oxidation state IV), had no effect oncell proliferation even at 250 μM (Table 5). These results demonstratethat the vanadium(IV)-containing bis(cyclopentadienyl)-metal complex hascytotoxic activity against human testicular cancer cells.

Sixteen structurally similar compounds with differing substituentsaround the ancillary position of the Cp2-vanadium(IV) unit were examinedfor their growth-inhibiting properties. These vanadocene complexesincluded: 4 vanadocene dihalides (VDB, VDC, VMDC, and VDI), 5 vanadocenedi-pseudohalides (VDA, VDCN, VDOCN, VDSCN, and VDSeCN), 3 vanadocenedisubstituted derivatives (VDT, VDCO, and VDFe), and 4 chelatedvanadocenes (VD(acac), VDH, VD(bpy), and VD(atc)). The cytotoxic effectsof these vanadocenes were tested at 7 different concentrations (1.9 μMto 250 μM). Each one of the 4 vanadocene dihalides, 5 vanadocenedi-pseudohalides, 3 vanadocene disubstituted derivatives, and 4 chelatedvanadocenes with various substituents covalently coordinated as ligandsto the central metal ion vanadium (IV) induced a concentration-dependentcytotoxicity to both Tera-2 and Ntera-2 cells at micromolarconcentrations. However, marked differences were noted in their potency.

The IC₅₀ values of VDB, VDC, VMDC, VDI, VDA, VDCN, VDOCN, VDSCN, VDSeCN,VDT, VDCO, VDFe, VD(acac), VDH, VD(BPY), and VD(DTC) calculated fromconcentration-response curves for the two cell lines are shown in Table5. The IC₅₀ values for the 16 vanadocenes evaluated ranged from 9 to 221μM. In general, vanadocenes were less potent than cisplatin (IC₅₀=˜5μM). However, the cytotoxic effects of the most potent vanadocenes,VDSCN and VDSeCN, were comparable to those of cisplatin (IC₅₀ values˜9-22 μM) when tested side-by-side under identical experimentalconditions. The variable potency of vanadocenes suggest that the variousmono and bidentate ligand groups affect the cytotoxic activity of thesecompounds. The potential cytotoxic effects of vanadium [vanadyl(IV)sulfate] were tested at the same concentrations. In sharp contrast tothe organometallic compounds containing vanadium(IV), inorganic vanadium(oxidation state IV) salt lacked cytotoxic activity even at 250 μM(Table 5). Importantly, the anticancer activity of these compounds wasnot restricted to testicular cancer cells. Both compounds also killedhuman glioblastoma cells at low micromolar concentrations (Table 5).

Vanadocenes Induce Apoptosis in Human Testicular Cancer Cells

In order to determine if the cytotoxicity of vanadocenes is associatedwith apoptotic cell death, Tera-2 and Ntera-2 cells were cultured withvanadocenes (100 μM) for 24 h and then subjected to flow cytometricanalysis for dUTP incorporation by the TdT-mediated TUNEL assay. FIGS. 2and 3 depict the two-color flow cytometric contour plots of cells fromrepresentative TUNEL assays. Control Tera-2 and Ntera-2 cells weretreated for 24 hours at 37° C. with 0.1% DMSO whereas test cells weretreated for 24 hours at 37° C. with a vanadocene compound at 100 μMfinal concentration. The TdT-dependent incorporation of FITC-dUTP wasdramatically increased in vanadocene-treated cells as a result ofabundance of free 3′-hydroxyl DNA ends created by endonuclease-mediatedDNA fragmentation. Among the 16 vanadocenes evaluated by the flowcytometric TUNEL assay, 15 caused a marked increase in TUNEL-positivenuclei ranging from 44.5% to 88% for Tera-2 cells and 38.7% to 99.6% forNtera-2 cells respectively (Table 5).

FIGS. 2 and 3 also depict the two-color confocal microscopy images ofDMSO treated control cells and vanadocene-treated test cells.Vanadocene-treated cells showed dual fluorescence, consistent withapoptosis. Furthermore, vanadocene-treated cells displayed thecharacteristic morphologic features of apoptotic cell death, includingcellular shrinkage, chromatin condensation, and the appearance oftypical apoptotic bodies. Apoptosis after vanadocene treatment was alsoevident from the concentration-dependent emergence of a hypodiploid(<2N) peak in the DNA histograms of PI-stained cells, which wasaccompanied by nonselective loss of G0/1, S, and G2M phase cells (FIG.4).

Cytotoxic Activity of Vanadocene Compounds Against Human Glioblastoma,Leukemia, and Breast Cancer Cell Lines

In MTT assays, both U373 glioblastoma cells (Table 5 and FIG. 5) andNALM-6 B-lineage acute lymphoblastic leukemia (ALL) cells (Table 5) werefound to be sensitive to the cytotoxic activity of vanadocenes. Similarto testicular cancer cells, glioblastoma cells were sensitive to VDC butnot to other metallocene dichlorides (Table 5). Interestingly, VDI andVD(dtc) were >1-log more active against U373 cells than they wereagainst TERA-2 or NTERA-2 cells. VDSeCN which was the most activevanadocene compound against testicular cancer cells was found to be themost active vanadocene compound against glioblastoma cells as well(Table 5, FIG. 5). Only 4 vanadocenes (VDC, VDB, VDI, VDA, VDSeCN) weretested against NALM-6 leukemia cells and all 4 were active and VDSeCNwas the most potent (Table 5).

To determine whether the cytotoxicity of vanadocenes againstnon-testicular cancer cells was also associated with apoptosis, MC540binding (as an early marker of apoptosis) and PI permeability (as amarker of advanced stage apoptosis) were simultaneously measured in HL60acute myeloid leukemia (AML), MDA-MB231 and BT-20 breast cancer, as wellas U87 glioblastoma cells 24 hours after exposure to the lead vanadocenecompound VDSeCN. As shown in FIG. 6, VDSeCN induced apoptosis in all 4cell lines in a concentration-dependent fashion. At 100 μM, 51% of HL-60cells, 67% of MDA-MB-231 cells, 40% of BT-20 cells, and 79% of U87 cellsshowed dual MC540/PI fluorescence consistent with advanced stageapoptosis. The total percentage of apoptotic cells (both early MC540⁺PI⁻and advanced stage MC540⁺PI⁺) at this concentration were 98% for HL-60cells, 72% for MDA-MB-231 cells, 61% for BT-20 cells, and 90% for U87cells (FIG. 6). Apoptosis after both VDSeCN and VDC treatment was alsoevident from the concentration-dependent emergence of a hypodiploid(<2N) peak in the DNA histograms of PI-stained HL-60 leukemia cells,which was accompanied by nonselective loss of G0/1, S, and G2M phasecells (FIG. 7).

Immunofluorescence staining with anti-α-tubulin antibody and the nucleardye toto-3 in combination with confocal laser scanning microscopy wasperformed to examine the morphological features of BT-20 breast cancercells treated with VDC. After 48-72 hr of exposure to 25 μM VDC, most ofthe BT-20 cells showed an abnormal architecture with complete disruptionof microtubules, marked shrinkage, nuclear fragmentation and inabilityto adhere to the substratum (FIG. 8). The cytotoxic effects ofmetallocene compounds were systematically assessed, including 16vanadocene diacido complexes against human testicular cancer cells.Vanadoces exhibited significant cytotoxicity against testicular cancercells and induced apoptosis within 24 hours. Vanadocenes withdithiocyanate (VDSCN) and diselenocyanate (VDSeCN) as ancillary ligandswere identified as the most potent cytotoxic compounds. Vanadocenes werecapable of inducing apoptosis not only in testicular cancer cells, butin ALL, AML, breast cancer, and glioblastoma cells as well. Thus, thepotent cytotoxic activity of vanadocene compounds was not limited totesticular cancer cells. The lead compound VDSeCN may be useful as ananti-cancer agent.

Among the five metallocenes evaluated, only vanadium (IV)-containingcomplexes exhibit cytotoxicity against human testicular cancer cells.Biological evaluation of a novel series of systematically coordinatedvanadocenes with dihalide, pseudodihalide, disubstituted derivatives,and chelated ancillary ligands demonstrated that the cytotoxic potencyof vanadocenes can be modulated by the coordinated ligands. All 16vanadocenes tested side-by-side induced apoptosis of testicular cancercells as shown by increased dUTP incorporation into nuclear DNA.Confocal microscopy images confirmed the results of dUTP incorporationin the nuclei of representative vanadocene-treated cells. In contrast,the inorganic vanadium(IV) compound, vanadyl sulfate, had no cytotoxiceffects on testicular cancer cells. Thus, although the cytotoxic effectof vanadocenes was primarily dependent upon the central vanadium(IV)ion, the two cyclopentadienyl units attached to vanadium(IV)coordination sites, and the various mono and bidentated ligand groupscoordinated to the bis(cyclopentadienyl)vanadium (IV) moiety appear tobe also very important for their anticancer activity.

Metallocene diacido complexes, especially titanocene dichloride andvanadocene dichloride may be useful as a chemopreventive agent and hasbeen found to be active against development of mammary, lung, colon,skin, as well as against various human tumors heterotransplanted toathymic mice. Köpf-Maier, P., Kopf, H. Tumor inhibition by titanocenecomplexes: activity against B16 melanoma and colon 38 carcinoma.Arzneim-Forsch/Drug Res., 37: 532-534, 1987. Moebus, V. J., Stein, R.,Kieback, D. G., Runnebaum, I. B., Sass, G., Kreienberg, R. Antitumoractivity of new organometallic compounds in human ovarian cancer celllines and comparison to platin derivatives. Anticancer Res., 17:815-822, 1997. Surprisingly, unlike vanadocenes, TDC as well as othernon-vanadium(IV)-containing metallocenes had no effect on the growth oftesticular cancer cells. Therefore, it is likely that the mechanism ofvanadocene-mediated growth inhibition is different from that induced bytitanocene or other metallocenes reported in other types of cancercells. Köpf-Maier, P., Kopf, H. Tumor inhibition by titanocenecomplexes: activity against B16 melanoma and colon 38 carcinoma.Arzneim-Forsch/Drug Res., 37: 532-534, 1987. Köpf-Maier, P. Tumorinhibition by titanocene complexes: influence upon two xenografted humanlung carcinomas. J. Cancer Res. Clin. Oncol., 113: 342-348, 1987.McLauglin, M. L., Cronan, J. M., Schaller, T. R., Snelling, R. D.DNA-metal binding by antitumor-active metallocene dichlorides frominductively coupled plasma spectroscopy analysis: Titanocene dichlorideforms DNA-Cp₂Ti or DNA-CpTi adducts depending on pH. J. Am. Chem. Soc.,112: 8949-8952, 1990. This difference observed between TDC and VDC ismost likely due to structural orientation as well as the one electronconfiguration of TDC when compared to vanadocenes such as VDC. Inaddition, unlike other transition metals, vanadium(IV)-containingmetallocenes can have pleiotropic effects in cells such as modulation ofthe cellular redox potential, Rehder, D. The bioinorganic chemistry ofvanadium. Angew Chem. Int. Ed. Engl., 30: 148-167, 1991, and Choukroun,R., Douziech, B., Pan, C., Dahan, F., Cassoux, P. Redox properties ofcationic vanadium(IV): [C_(p2)VCH₃(CH₃CN)][BPh₄]. Oraganometallics, 14:4471-4473, 1995, increased phosphorylation, Heffetz, D., Bushkin, I.,Dror, R., Zick, Y. The insulinomimetic agents H202 and vanadatestimulate protein tyrosine phosphorylation in intact cells. J. Biol.Chem., 265: 2896-2902, 1990. Stem, A., Yin, X., Tsang, S. S., Davison,A., Moon, J. Vanadium as a modulator of cellular regulatory cascades andoncogene expression. Biochem. Cell Biol., 71: 103-112, 1993, andgeneration of reactive oxygen intermediates. Byczkowski, J. Z., Wan, J.Z., Kulkarni, A. P. Vanadium-mediated lipid peroxidation in microsomesfrom human term placenta. Bull. Environ. Contain. Toxicol., 41: 696-703,1988. Ozawa, T., Hanaki, A. ESR evidence for the formation of hydroxylradicals during the reaction of vanadyl ions with hydrogen peroxide.Chem. Pharm. Bull., 37: 1407-1409, 1989. Carmichael, A. J.Vanadyl-induced Fenton like reaction in RNA: an ESR and spin trappingstudy. FEBS Lett., 261: 165-170, 1990. Younes, M., Strubelt, O.Vanadate-induced toxicity towards isolated perfused rat livers: The roleof lipid peroxidation. Toxicology, 66: 63-74, 1991. Keller, J., Sharma,R. P., Grover, T. A., Piette, L. H. Vanadium and lipid peroxidation:Evidence of involvement of vanadyl and hydroxyl radical. Arch. Biochem.Biophys. 265: 524-533, 1988. Sakurai, H., Nakai, M., Miki, T., Tsuchiya,K., Takada, J., Matsushita, R. DNA cleavage by hydroxyl radicalsgenerated in a vanadyl ion-hydrogen peroxide system. Biochem. Biophys.Res. Commun. 189: 1090-1095, 1992. Sakurai, H., Tamura, H., Okatani, K.Mechanism for a new antitumor vanadium complex hydroxylradical-dependent DNA-cleavage by 1,10-phenanthroline-vanadyl complex inthe presence of hydrogen peroxide. Biochem. Biophys. Res. Commun., 206:133-137, 1995. Shi, X., Wang, P., Jiang, H., Mao, Y., Ahmed, N., Dalal,N. Vanadium(IV) causes 2′-deoxyguanosine hydroxylation anddeoxyribonucleic acid damage via free radical reactions. Ann. Clin. Lab.Sci. 26: 39-49, 1996.

The potential therapeutic applications of organovanadium compoundsincluding vanadocenes in vivo, particularly to suppress tumor cellgrowth via apoptosis, reduce hyperlipidemia, and hypertension viatyrosine kinase-signalling pathways with relatively few adverse effectshas sparked interest as a new class of pharmacological agents.Eliopoulos, A. G., Kerr, D. J., Maurer, H. R., Hilgard, P., Spandidos,D. A. Induction of the myc but not CHras promoter by platinum compounds.Biochem. Pharmacol. 50: 33-38, 1995. Orvig, C., Thompson, K. H., Battel,M., McNeill, J. H. Vanadium compounds as insulin mimetics. In: Sigel H,Sigel A, eds. Metal ions in biological systems. New York:Marcel Dekker31: 595-616, 1995. Tsiani, E., Fantus, I. G. Vanadium compounds:biological actions and potential as pharmacological agents. TrendsEndocrinol. Metab. 8: 51-58, 1997. Nechay, B. R. Mechanisms of action ofvanadium. Annu. Rev. Pharmacol. Toxicol. 24: 501-524, 1984. Theapoptosis-inducing cytotoxic effects of novel vanadocenes against humancancer cells reported herein indicate that vanadocenes may be useful asanticancer agents.

TABLE 1 Vanadocene Compounds — Type 1 Series UV-vis [λ (nm); IR SpectralData Elemental Analysis # Compound Chemical Structure Solvent] [cm⁻¹][Found (Calcd.)] 1 HDC

312, 268, 232 (CH₂Cl₂) 3105(vs), 1439(vs), 1365(m), 1126(m), 1014(vs),920(s,d), 802(vs), 816(vs), 611(m) C, 32.09 (31.62) H, 2.75 (2.63) Cl,18.98 (18.71) 2 MDC

678, 436, 299, 283, 267 (DMSO) 3093(vs), 1420(vs), 1375(m), 1100(m),1060(m), 825(vs), 590(m) C, 40.8 (40.4) H, 3.39 (3.37) Cl, 24.4 (23.9) 3TDC

526, 391, 314, 255 (CH₂Cl₂) 3105(vs), 1441(vs), 1368(m), 1130(m),1016(vs), 956(m), 872(s), 820(s) C, 48.56 (48.2) H, 4.03 (4.01) Cl,28.78 (28.5) 4 ZDC

341, 294, 236 (CH₂Cl₂) 3104(vs), 1435(s), 1363(m), 1122(s), 1014(s),815(s), 610(m) C, 41.01 (41.09) H, 3.4 (3.4) Cl, 24.84 (24.31) 5 VDC

767, 647, 380, 283, 244 (CH₂Cl₂) 3095(vs), 1444(s), 1433(s), 1130(m),1070(m), 887(m), 825(vs) C, 47.88 (47.62) H, 4.04 (3.97) Cl, 27.64(28.1)

TABLE 2 Vanadocene Compounds — Type 2 Series UV-vis [λ (nm); IR SpectralData Elemental Analysis # Compound Chemical Structure Solvent] [cm⁻¹][Found (Calcd.)] 6 VDB

733, 412, 298, 232 (CH₂Cl₂) 3089(vs), 1425(s), 1431(m), 1373(m),1363(w), 1128(w), 1024(m), 1014(m), 825(vs) C, 35.19 (35.19) H, 2.9(2.92) Br, 46.91 (46.92) 7 VDI

620, 552, 352, 296, 232 (CH₂Cl₂) 3095(s), 1425(s), 1373(m), 1182(m),1024(m), 1014(m), 825(vs) C, 28.1 (27.58) H, 2.42(2.3) I, 58.4 (58.9) 8VDA

434, 314, 257, 233 (CH₂Cl₂) 3114(vs), 1448(m), 1375(m), 1126(w),1080(s), 1024(m), 835(vs), 590(m) C, 45.28 (45.28) H, 3.73 (3.77) N,31.16 (31.2) 9 VDN

605, 394, 307, 250 (CH₂Cl₂) 3114(vs), 2120(s), 2110(s), 1435(s),1420(s), 1126(m), 1014(s), 881(s), 848(s), 845(s) C, 60.98 (61.89) H,4.30 (4.29) N, 11.45 (12.00) 10  VDO

742, 373, 277, 237 (CH₂Cl₂) 3531(m), 3110(m), 2248(vs), 2217(vs),1444(s), 1330(s), 1024(w), 833(vs), 603(s), 593(s) C, 53.85 (54.3) H,3.97 (3.8) N, 10.2 (10.6) 11  VDOCN

710, 490, 257, 227 (CH₂Cl₂) 3110(m), 2657(w), 2117(vs), 1444(m),1330(s), 1261(w), 1018(m), 950(m), 833(vs), 635(vs), 424(w) C, 51.35(51.06) H, 3.97 (3.87) N, 5.65 (5.41) Cl, 13.45 (13.73) 12  VDS

739, 463, 401, 270, 251 (CH₂Cl₂) 3087(s), 2086(vs), 2067(vs), 1433(s),1423(m), 1010(m), 840(vs), 480(vw) C, 47.55 (47.05) H, 3.26 (3.59) S,20.91 (20.91) 13  VDSe

716, 488, 456, 270, 251 (CH₂Cl₂) 3076(s), 2085(vs), 2065(vs), 1444(m),1431(s), 1074(w), 1008(m), 962(m), 843(vs) C, 36.85 (36.83) H, 2.64(2.56) N, 6.97 (7.1) 14  VDFe

648, 575, 362, 311, 265, 240 (CH₂Cl₂) 3109(m), 2924(m), 2318(s),2289(m), 1622(m), 1447(s), 1435(m), 1358(w), 1027(s), 1012(s), 856(s),846(s) C, 31.2 (31.6) H, 2.56 (2.9) N, 3.48 (3.1) Cl, 40.31 (39.98) 15 VDT

740, 640, 370, 309, 270, 230 (CH₂Cl₂) 3118(s), 1564(vs), 1440(s),1350(s), 1218(s), 1194(w), 1149(vs), 1032(s), 959(w), 843(vs), 638(vs)C, 44.81 (44.45) H, 3.99 (3.96) S, 7.52 (7.46)

TABLE 3 Vanadocene Compounds — Type 3 Series UV-vis [λ (nm); IR SpectralData Elemental Analysis # Compound Chemical Structure Solvent] [cm⁻¹][Found (Calcd.)] 16 VD(acac)

740, 640, 370, 309, 270, 230 (CH₂Cl₂) 3118(s), 2295(w), 1564(vs),1440(s), 1350(s), 1267(sb), 1218(s), 1194(w), 1149(vs), 1032(s), 983(w),910(w), 843(s), 573(s) C, 44.81 (44.45) H, 3.99 (3.96) S, 7.52 (7.46) 17VD(hƒ-acac)

575, 377, 314, 271, 244 (CH₂Cl₂) 3117(m), 1637(vs), 1597(w), 1552(w),1446(s), 1260(vs), 1219(vs), 1163(vs), 1142(s), 1120(m), 1030(vs),851(s), 640(vs) C, 35.76 (35.89) H, 2.08 (2.06) S, 5.89 (5.98) 18VD(bpy)

780, 326, 272, 241 (CH₂Cl₂) 3135(m), 3099(s), 1605(s), 1504(m), 1477(m),1452(s), 1437(vs), 1307(m), 1257(vs), 1232(vs), 1028(vs), 862(vs),771(vs), 636(vs) C, 52.48 (53.1) H, 3.72 (3.69) N, 2.51 (2.58) S, 5.73(5.9) 19 VD(cat)

711, 438, 337, 292, 275, 259 (CH₂Cl₂) 3100(w), 3080(w), 2951(m),2945(w), 2860(w), 1468(s), 1438(m), 1404(m), 1359(w), 1261(vs), 1012(w),804(vs), 638(w) C, 66.79 (66.45) H, 4.93 (4.88) 20 VD(dtc)

621, 535, 392, 330, 276, 270, 230 (CH₃CN) 3107(m), 1632(s), 1595(w),1538(w), 1439(s), 1212(s), 1201(s), 1156(s), 1123(m), 1019(s), 855(s),641(s) C, 43.41 (43.31) H, 4.14 (4.18) N, 2.86 (2.93) S, 19.98 (20.08)21 VDPH

680, 501, 377, 314, 261, 233 (CH₂Cl₂) 3117(s), 1600(m), 1539(s),1495(m), 1450(m), 1300(m), 1281(s), 1244(s), 1173(s), 999(m), 758(m),694(m), 638(s) C, 36.85 (36.83) H, 2.64 (2.56) N, 6.97 (7.1) 22 VDH

710, 550, 401, 300, 261, 233 (CH₂Cl₂) 1695(mb), 1635(m), 1500(vs),1450(s), 1280(s), 1260(s), 1215(vs) 1144(s), 959(m), 758(m), 635(m),540(w), 480(m) C, 38.12 (38.61) H, 3.72 (3.46) N, 3.26 (3.46)

TABLE 4 Vanadocene Compounds — Type 4 Series UV-vis [λ (nm); IR SpectralData Elemental Analysis # Compound Chemical Structure Solvent] [cm⁻¹][Found (Calcd.)] 23 VMDC

760, 659, 383, 286, 233 (CH₂Cl₂) 3135(m), 3099(s), 1307(m), 1028(s),862(s), 771(s), 636(s) C, 49.92 (49.82) H, 4.90 (5.19) Cl, 24.90 (24.60)24 VPMDC

740, 652, 440 (CHCl₃) 3118(s), 1194(w), 1149(m), 1032(s), 959(w),843(vs), 638(vs) C, 59.9 (61.2) H, 7.5 (7.6) Cl, 13.1 (13.0) 25 VPMOC

680, 605, 290, 250 (CHCl₃) 2962(m), 2906(s), 2858(m), 1621(w), 1497(m),1437(s), 1375(vs), 1066(s), 1014(m), 960(m), 943(m), 806(m), 744(m),700(m) C, 43.29 (43.98) H, 5.62 (5.54) Cl, 27.54 (27.67)

TABLE 5 In Vitro Cytotoxic Activity of Vanadocene Compounds AgainstHuman Cancer Cells. IC₅₀[MTT](μM) % Apoptosis at 100 μM Compound TERA-2NTERA-2 U373 NALM-6 TERA-2 NTERA-2 Metallocene DichloridesHDC >250 >250 >250 N.D. N.D. N.D. MDC >250 >250 >250 N.D. N.D. N.D.TDC >250 >250 >250 N.D. N.D. N.D. ZDC >250 >250 >250 N.D. N.D. N.D. VDC81 74    42 19 88 (87,89) 35 (34, 34) Vanadocene Diacido Compounds VDB154 70    62 17 84 (83, 86) 93 (93, 94) VDI 221 204    5 18 72 (71, 73)57 (46, 69) VDA 70 68    51 16 85 (78, 92) 85 (78, 93) VDCO 50 90 N.D.N.D. 73 (67, 78) 98 (97, 99) VDCN 51 93    53 N.D. 80 (78, 82) 97 (96,97) VDOCN 31 63 N.D. N.D. 85 (80, 90) 95 (94, 96) VDSCN 23 17    19 N.D.64 (58, 69) 53 (46, 60) VDSeCN 9 22    2  3 78 (75, 80) 99.6 ± 0.2 (N =4) VDFe 100 113 N.D. N.D. 85 (84, 87) 87 (81, 94) VDT 76 137 N.D. N.D.71 (71, 71) 55 ± 26 (N = 4) Vanadocene Chelated Compounds VD (acac) 6475 N.D. N.D. 72 ± 17 48 ± 13 (N = 3) VD (bpy) 37 53 N.D. N.D. N.D. 78(77, 78) VD (dtc) 60 83    7 N.D. 83 (82, 84) 67 ± 13 (N = 3) VD (cat)79 93 N.D. N.D. N.D. 12 (9, 15) VDPH >250 61 N.D. N.D. 88 ± 3 (N = 3) 90± 3 (N = 4) VDH 118 125 >250 N.D. 84 ± 2 (N = 3) 20 ± 5 (N = 3)Substituted Cyclopentadienyl Compounds VMDC 123 86    61 N.D. 45 (27,61) 13 (13, 14) VPMDC 127 44   172 N.D. 35 (35, 36) 34 (33, 35)VPMOC >250 77 N.D. N.D.  5 (5, 6)  6 (6, 7) Control VDSO₄ >250 >250 N.D.N.D. N.D.

Example 2 Oxovanadium (IV) Compounds

Materials and Methods

The oxovanadium (IV) complexes were synthesized based on previouslypublished chemistry of VO(phen) and VO(phen)₂ complexes. Sakurai, et.al, Biochemical and Biophysical Research Communications, Vol. 206, No.1, (1995). Selbin, et. al, Chemical Reviews, Vol. 65, No. 2 (1965).Briefly, these complexes were synthesized by reacting an aqueoussolution of vanadyl sulfate with an ethanol solution or a chloroformsolution of the ligands.

The complexes purified from chloroform, ether and/or water werecharacterized by Fourier transform infrared spectroscopy (FT-Nicoletmodel Protege 460; Nicolet Instrument Corp., Madison, Wis.), UV-visiblespectroscopy (DU 7400 spectophotometer; Beckman Instruments, Fullerton,Calif.) and elemental analysis (Atlantic Microlab, Inc., Norcross, Ga.).These oxovanadium (IV) complexes have an octahedral or square pyramidalgeometry with the oxo ligand (O²⁻) in the axial site. The oxovanadiumcomplexes are stabilized with bidentate ligands which form a 5-memberedring with the vanadium atom. The choice of these three organic ligands(phenanthroline, bipyridyl, bipyrimidal and acetophenone) was based onthe reported fact that the cationic oxovanadium(IV) complex ofphenanthroline is superior to cisplatin(cis-diamminedichloroplatinum[II]) with respect to antitumor activity,the structural similarity of bipyridyl ring to phenanthroline, as wellas the neutral nature of acetophenone complex of oxovanadium(IV).

Structural variations of the ligands included addition of bromo, chloroor methyl groups on the phenanthroline, bipyridyl or acetophenone rings.The chemical structures of the oxovanadium (IV) complexes, including 8complexes with 1,10-phenanthroline and 4 complexes with 2,2′-bipyridyl,and one neutral complex, bis-5′-bromo-2′-hydroxyacetophenone, aredepicted in Table 6. The synthesis and analysis of the compounds can besummarized as follows.

[VO(Phen)(H₂O)₂](SO₄) (Phen=1,10-phenanthroline)(diaqua)(1,10-phenanthroline)oxovanadium(IV) sulfate (Compound 26). Achloroform solution of 1,10-phenanthroline (90.1 mg, 0.5 mmol) was addedto VO(SO₄).3H₂O (108.5 mg, 0.5 mmol) in 6 mL of water. The resultinggreen solution was stirred at room temperature for 3 h, and then storedin refrigerator for one day. The green water layer was separated fromthe chloroform layer, and water was removed by vacuum. The green solidproduct (165 mg, 87%) was washed with chloroform and ether, and dried inair. Anal. Calcd for [VO(Phen)(H₂O)₂](SO₄)(C₁₂H₁₂N₂O₇SV): C, 38.01; H,3.20; N, 7.39. Found: C, 38.57; H, 3.07; N, 7.47. IR spectrum: ν(V=O)978 cm⁻¹.

[VO(SO₄)(Phen)₂]bis(1,10-phenanthroline)sulfatooxovanadium(IV) (Compound27). An ethanol solution of 1,10-phenanthroline (180.2 mg, 1.0 mmol) wasadded to VO(SO₄).3H₂O (108.5 mg, 0.5 mmol) in 6 mL of water. Theresulting brown solution was stirred at room temperature for 3 h, andbrown microcrystals precipitated from the solution upon standing at −20°C. The product (247 mg, 89%) was washed with chloroform and ether, anddried in air. Anal. Calcd for [VO(SO₄)(Phen)₂].2H₂O (C₂₄H₂₀N₄O₇SV): C,51.53; H, 3.60; N, 10.01. Found: C, 50.92; H, 3.65; N, 9.87. IRspectrum: ν(V=O) 978 cm⁻¹.

[VO(Me₂-Phen)(H₂O)₂](SO₄)(Me₂-Phen=4,7-dimethyl-1,10-phenanthroline)(diaqua)(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate(Compound 28). A chloroform solution of 4,7-dimethyl-1,10-phenanthroline(104.1, 0.5 mmol) was added to VO(SO₄).3H₂O (108.5 mg, 0.5 mmol) in 6 mLof water resulting in the precipitation of a green solid. The reactionmixture was stirred at room Temperature for one day, and the product(128 mg, 63%) was filtered and washed with water and ether, and dried inair. Anal. Calcd for [VO(Me₂-Phen)(H₂O)₂](SO₄)(C₁₄H₁₆N₂O₇SV): C, 41.29;H, 3.96; N, 6.88. Found: C, 41.08; H, 4.12; N, 6.74. IR spectrum:ν)(V=O) 978 cm⁻¹.

[VO(SO₄)(Me₂-Phen)₂]bis(4,7-dimethyl-1,10-phenanthroline)sulfatooxovanadium(IV) (Compound 29) was prepared by mixing an aqueoussolution of VO(SO₄).3H₂O (54.3 mg, 0.25 mmol) with an ethanol solutionof 4,7-dimethyl-1,10-phenanthroline (104.2 mg, 0.5 mmol) at roomtemperature. The reaction mixture was stirred at room temperature for 2days. During this time, the blue solution first turned to green and thento brown. The brown solid product (107 mg, 68%) was obtained by removingsolvent and washing with chloroform and ether, and drying under vacuum.Anal. Calcd for [VO(SO₄)(Me₂-Phen)₂].3H₂O (C₂₈H₃₀N₄O₈SV): C, 53.08; H,4.77; N, 8.84. Found: C, 53.01; H, 4.58; N, 8.84. IR spectrum: ν(V=O)973 cm⁻¹.

[VO(Cl-Phen)(H₂O)₂](SO₄)(diaqua)(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate (Compound 30) was prepared by mixing an aqueoussolution of VO(SO₄).3H₂O (108.5 mg, 0.5 mmol) with an ethanol solutionof 5-chloro-1,10-phenanthroline (107.3 mg, 0.5 mmol) at roomtemperature. The reaction mixture was stirred at room temperature fortwo days. During this time, the blue solution turned to green. The greensolid product (142.3 mg, 69%) was obtained by removing solvent andpurifying from water and ether, and drying under vacuum. Anal. Calcd for[VO(Cl-Phen)(H₂O)₂](SO₄) (C₁₂H₁₁N₂O₇ClSV): C, 34.84; H, 2.68; N, 6.77.Found: C, 34.96; H, 2.64; N, 6.84. IR spectrum: ν(V=O) 964 cm⁻¹.

[VO(SO₄)(Cl-Phen)₂](Cl-Phen=5-chloro-1,10-phenanthroline)bis(5-chloro-1,10-phenanthroline)sulfatooxovanadium(IV)(Compound 31). An ethanol solution of 5-chloro-1,10-phenanthroline(214.7 mg, 1.0 mmol) was added to VO(SO₄).3H₂O (108.5 mg, 0.5 mmol) in 6mL of water. The resulting brown solution was stirred at roomtemperature for 7 h, and a small amount of brown microcrystalsprecipitated upon standing at −20° C. The brown solid product (222 mg,71%) was obtained by removing solvent and washing with chloroform andether, and drying under vacuum. Anal. Calcd for [VO(SO₄)(Cl-Phen)₂].2H₂O(C₂₄H₁₈N₄O₇Cl₂SV): C, 45.88; H, 2.89; N, 8.92. Found: C, 45.44; H, 2.87;N, 8.75. IR spectrum: ν(V=O) 962 cm⁻¹.

[VO(NO₂-Phen)(H₂O)₂](SO₄)(NO₂-Phen=5-nitro-1,10-phenanthroline)(diaqua)(5-nitro-1,10-phenanthroline)oxovanadium(IV) sulfate (Compound32) was prepared by adding a blue aqueous solution of VO(SO₄).3H₂O(108.5 mg, 0.5 mmol) into a yellow suspension of5-nitro-1,10-phenanthroline (112.6 mg, 0.5 mmol) in ethanol at roomtemperature. The reaction mixture turned to green solution immediately,and then was stirred at room temperature for two days. During this time,some brown solid precipitated. The brown solid was filtered off and thefiltrate was evaporated to dryness to give rise to a green solid. Thegreen solid product (90.7 mg, 43%) was obtained by purifying from waterand ether, and drying under vacuum. Anal. Calcd for[VO(NO₂-Phen)(H₂O)₂](SO₄) (C₁₂H₁₁N₃O₉SV): C, 33.97; H, 2.61; N, 9.91.Found: C, 33.70; H, 2.54; N, 9.78. IR spectrum: ν(V=O) 980 cm⁻¹.

[VO(SO₄)(NO₂-Phen)₂]bis(5-nitro-1,10-phenanthroline)sulfatooxovanadium(IV)(Compound 33) was prepared by mixing a blue aqueous solution ofVO(SO₄).3 H₂O (108.5 mg, 0.5 mmol) with a yellow suspension of5-nitro-1,10-phenanthroline (225.2 mg, 1 mmol) in ethanol at roomtemperature. A brown solution was generated upon the mixing and a yellowsolid precipitated gradually. The reaction mixture was stirred at roomtemperature for one day. The yellow solid product (180 mg, 54%) wasobtained by filtration and washing with water, chloroform and ether, anddrying in air. Anal. Calcd for [VO(SO₄)(NO₂-Phen)₂].3H₂O(C₂₄H₂₀N₆O₁₂SV): C, 43.18; H, 3.02; N, 12.59. Found: C, 42.61; H, 2.98;N, 12.28. IR spectrum: ν(V=O) 976 cm⁻¹.

[VO(Bpy)(H₂O)₂](SO₄)(Bpy=2,2′-bipyridine)(diaqua)(2,2′ bipyridyl)oxovanadium(IV) sulfate (Compound 34). A chloroform solution of2,2′-bipyridine (78.1 mg, 0.5 mmol) was added to VO(SO₄).3H₂O (108.5 mg,0.5 mmol) in 6 mL of water. The resulting green solution was stirred atroom temperature overnight, and then the water layer was separated fromthe chloroform layer. The green solid product (105 mg, 59%) was obtainedby removing water and washing with ether, and drying in air. Anal. Calcdfor [VO(Bpy)(H₂O)₂](SO₄) (C₁₀H₁₂N₂O₇SV): C, 33.81; H, 3.41; N, 7.89.Found: C, 33.77; H, 3.16; N, 7.72. IR spectrum: ν(V=O) 978 cm⁻¹.

[VO(SO₄)(BPY)₂]bis(2,2′-bipyridyl)sulfatooxovanadium(IV) (Compound 35).An ethanol solution of 2,2′-bipyridine (156.2 mg, 1.0 mmol) was added toVO(SO₄).3H₂O (108.5 mg, 0.5 mmol) in 6 mL of water. The resulting brownsolution was stirred at room temperature overnight, and then stored at−20° C. for one day. The brown solid product (180 mg, 76%) was obtainedby removing solvent and washing with chloroform, ethanol and ether, anddrying in air. Anal. Calcd for [VO(SO₄)(Bpy)₂].H₂O (C₂₀H₁₈N₄O₆SV): C,48.69; H, 3.68; N, 11.36. Found: C, 48.38; H, 3.81; N, 11.48. IRspectrum: ν(V=O) 978 cm⁻¹.

[VO(Me₂-bpy)(H₂O)₂](SO₄)(Me₂-bpy=4,4′-dimethyl-2,2′-bipyridyl)(diaqua)(4,4′-dimethyl-2,2′-bipyridyl)oxovanadium(IV) sulfate (Compound36). A chloroform solution of 4,4′-dimethyl-2,2′-bipyridyl (104.1 mg,0.5 mmol) was added to VO(SO₄).3H₂O (108.5 mg, 0.5 mmol) in 6 mL ofwater. The reaction mixture was stirred at room temperature for one day,during this time a green solid formed. The product (130 mg, 68%) wasfiltered and washed with water and ether, and dried in air. Anal. Calcdfor [VO(Me₂-bpy)(H₂O)₂](SO₄)(C₁₂H₁₆N₂O₇SV): C, 37.61; H, 4.21; N, 7.31.Found: C, 37.49; H, 4.27; N, 7.16. IR spectrum: ν(V=O) 983 cm⁻¹.

[VO(SO₄)(Me₂-bpy)₂]bis(4,4′-dimethyl-2,2′-bipyridyl)sulfatooxovanadium(IV)(Compound 37). An ethanol solution of 4,4′-dimethyl-2,2′-bipyridyl (92.1mg, 0.5 mmol) was added to VO(SO₄).3H₂O (54.3 mg, 0.25 mmol) in 6 mL ofwater. The reaction mixture was stirred at room temperature for 20 h,and a small amount of green solid precipitated. The green solid wasremoved by filtration. The yellow solid product (72 mg, 51%) wasobtained by removing solvent and washing by chloroform and ether, anddrying under vacuum. Anal. Calcd for [VO(SO₄)(Me₂-bpy)₂].2H₂O(C₂₄H₂₈N₄O₇SV): C, 50.79; H, 4.97; N, 9.87. Found: C, 50.19; H, 4.81; N,9.67. IR spectrum: ν(V=O) 978 cm⁻¹.

[VO(Bipym)(H₂O)₂](SO₄)(Bipym=2,2′-bipyrimidine)(diaqua)(2,2′-bipyrimidine) oxovanadium(IV)sulfate (Compound 38) was prepared by mixing an aqueous solution ofVO(SO₄).3H₂O (86.8 mg, 0.4 mmol) with an ethanol solution of2,2′-bipyrimidine (63.3 mg, 0.4 mmol) at room temperature. The reactionmixture was stirred at room temperature for two days. During this time,the blue solution turned to green. The green solid product (112.3 mg,77%) was obtained by removing solvent and washing with ether, and dryingunder vacuum. Anal. Calcd for [VO(Bipym)(H₂O)₂](SO₄).0.5H₂O(C₈H₁₁N₄O_(7.5)SV): C, 26.24; H, 3.03; N, 15.30. Found: C, 25.99; H,2.91; N, 15.20. IR spectrum: ν(V=O) 978 cm⁻¹.

[VO(SO₄)(Bipym)₂]bis(2,2′-bipyrimidine)sulfatooxovanadium(IV) (Compound39) was prepared by mixing an aqueous solution of VO(SO₄).3H₂O (43.4 mg,0.2 mmol) with an ethanol solution of 2,2′-bipyrimidine (63.3 mg, 0.4mmol) at room temperature. The reaction mixture was stirred at roomtemperature for 5 days, and then kept in refrigerator overnight. Duringthis time, the blue solution turned to green. The green solid product(80 mg, 75%) was obtained by removing solvent and washing withchloroform and ether, and drying under vacuum. Anal. Calcd for[VO(SO₄)(Bipym)₂].3H₂O (C₁₆H₁₈N₈O₈SV): C, 36.03; H, 3.40; N, 21.01.Found: C, 35.83; H, 3.12; N, 21.12. IR spectrum: ν(V=O) 974 cm⁻¹.

[VO(Br,OH-acph)₂](Br,OH-acph=5′-bromo-2′-hydroxyacetophenone)bis(5′-bromo-2′-hydroxyacetophenone)oxovanadium(IV)(Compound 40). An ethanol solution of 5′-bromo-2′-hydroxyacetophenone(107.6 mg, 0.5 mmol) was added to VO(SO₄).3H₂O (108.5 mg, 0.5 mmol) inwater (5 mL). The solution turned to green immediately and a white solidprecipitated in a few minutes. After one hour, an aqueous solution ofNaOH (20 mg, 0.5 mmol) was added, and the reaction mixture was stirredat room temperature for one day. The resulting yellow solid product(63.5 mg, 40%) was filtered and washed with water and ether, and driedin air. Anal. Calcd for [VO(Br,OH-acph)₂].0.5H₂O (C₁₆H₁₃O_(5.5)Br₂V): C,38.13; H, 2.60; Br, 31.71. Found: C, 38.18; H, 2.36, Br, 31.65. IRSpectrum: ν(V=O) 971 cm⁻¹.

TABLE 6 Oxovanadium(IV) Compounds UV-vis IR Elemental Analysis λ, nm (ε,M⁻¹ cm⁻¹) υV = O, Found (Calcd.) Compound Structure [Solvent] cm⁻¹ C, H,N 26

970(sh), 745(34), 529(21), 432(sh) [H₂O] 978 38.57 (38.01), 3.07 (3.20),7.47 (7.39) 27

712(33), 540(sh), 436(sh) [H₂O] 978 50.92 (51.53), 3.65 (3.60), 9.87(10.01) 28

ND 978 41.08 (41.29), 4.12 (3.96), 6.74 (6.88) 29

664(83), [H₂O] 765(37), 448(236) [DMSO] 973 53.01 (53.08), 4.58 (4.77),8.84 (8.84) 30

745(30), 523(18), 438(sh), [H₂O] 964 34.96 (34.84), 2.64 (2.68), 6.84(6.77) 31

685(58), 535(sh), 446(sh), [H₂O] 962 45.44 (45.88), 2.87 (2.89), 8.75(8.92) 32

746(34), 444(sh) [H₂O] 980 33.70 (33.97), 2.54 (2.61), 9.78 (9.91) 33

757(34), 459(sh) [DMSO] 976 42.61 (43.18), 2.98 (3.02), 12.28 (12.59) 34

968(sh), 749(23), 535(sh), [H₂O] 978 33.77 (33.81), 3.16 (3.41), 7.72(7.89) 35

726(27), 533(19), 453(sh), [H₂O] 978 48.43 (48.69), 3.81 (3.68), 11.48(11.36) 36

ND 983 37.49 (37.61), 4.27 (4.21), 7.16 (7.31) 37

701(51), 535(49) [H₂O] 978 50.19 (50.79), 4.81 (4.97), 9.67 (9.87) 38

752(23), 572(sh), 406(sh), [H₂O] 978 25.99 (26.24), 2.91 (3.03), 15.20(15.30) 39

753(23), 564(sh), 410(sh), [H₂O] 974 35.83 (36.03), 3.12 (3.40), 21.12(21.01) 40

833(55), 621(62), 502(185) [DMSO] 971 C, 38.18 (38.13), H, 2.36 (2.60),Br, 31.65 (31.71)

Cell Lines and Culture Conditions

Human testicular cancer cell lines, Tera-2 (embryonal carcinoma) andNtera-2 (pluipotent embryonal carcinoma) were obtained from the AmericanType Culture Collection (ATCC) (Rockeville, Md.) and propagated in T-25,T-75, or T-150 cm² tissue culture flasks (Coming Corp., Corning, N.Y.)in McCoy's 5 A medium and Dulbecco's modified Eagle's mediumrespectively. Both media were supplemented with 10% fetal calf serum(FCS), 4 mM glutamine, 100 U/ml penicillin G, and 100 mg/ml streptomycinsulfate. All tissue culture reagents were obtained from LifeTechnologies Inc. (GIBCO-BRL), Gaithersburg, Md. Cell lines werecultivated for a minimum of two passages after thawing prior toexperimentation. Other cell lines that were used in this study were thehuman B-lineage acute lymnphoblastic leukemia (ALL) cell line NALM-6.Uckun, F. M., Evans, W. E., Forsyth, C. J., Waddick, K. G.,Tuel-Ahlgren, L., Chelstrom, L. M., Burkhardt, A., Bolen, J., Myers, D.E. Biotherapy of B-cell precursor leukemia by targeting genistein toCD19-associated tyrosine kinase. Science (Washington D.C.) 267:886-91,1995; T-lineage ALL cell line MOLT-3, Waurzyniak, B., Shneider, E. A.,Tumer, N., Yanishevski, Y., Gunther, R., Chelstrom, L., Wendorf, H.,Myers, D. E., Irvin, J. D., Messinger, Y., Ek, O., Zeren, T.,Chandan-Langlie, M., Evans, W. E., Uckun, F. M. In vivo toxicity,pharmacokinetics, and antileukemic activity of TXU (anti-CD7)-pokeweedantiviral protein immunotoxin. Clinical Cancer Research 3:881-890, 1997;AML cell line HL-60, Perentesis, J. P., Waddick, K. G., Bendel, A. E.,Shao, Y., Warman, B. E., Chandan-Langlie, M., and Uckun, F. M. Inductionof apoptosis in multi-drug resistant and radiation-resistant acutemyeloid leukemia cells by a recombinant fusion toxin directed againstthe granulocyte macrophage colony stimulating factor receptor. Clin.Cancer Res. 3:347-355, 1997; breast cancer cell lines MDA-MB-231 andBT-20, Uckun, F. M., Narla, R. K., Jun, X., Zeren, T., Venkatachalam,T., Waddick, K. G., Rostostev, A., Myers, D. E. Cytotoxic activity ofEGF-genstein against breast cancer cells. Clin. Cancer Res. 4:901-912,1998; glioblastoma cell lines U87 and U373, Narla, R. K., Liu, X.,Myers, D. E., and Uckun, F. M.4-(3′-Bromo-4′hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154):A novel quinazoline derivative with potent cytotoxic activity againsthuman glioblastoma cells. Clin. Cancer Res. 4:1405-1414, 1998.

MTT Assays.

MTT (3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazoliumbromide)-based colorimetric assays were used for evaluation of thecytotoxicity of vanadocene compounds. Mosmann, T. Rapid colorimetricassay for cellular growth and survival: application to proliferation andcytotoxicity assays. J. Immunol. Methods, 65: 55-63, 1983. Briefly,cells were harvested with 0.125% (w/v) trypsin-0.02% EDTA (GIBCO-BRL)from exponential-phase maintenance cultures and centrifuged (300 g×5min). After suspension and counting, cells were dispensed withintriplicate 96-well tissue culture plates in 100 μl volumes. After 24 hincubation, the culture medium was discarded and replaced with 100 μl offresh medium containing serial two-fold dilutions of drugs in medium toyield 1.9 μM to 250 μM. All compounds were reconstituted in DMSO to aconcentration of 100 mM, and the stock solution was made fresh for eachexperiment. Control wells consisted of medium containing 0.25% of DMSOalone were used for solvent control. Culture plates were then incubatedfor 24 h before adding 10 μl of MTT solution (5 mg/ml in PBS) to eachwell. Wells containing only medium and MTT were used as controls foreach plate. The tetrazolium/formazan reaction was allowed to proceed for4 h at 37° C., and then 100 μl of the solubilization buffer (10% sodiumdodecyl sulfate in 0.1% HCl) were added to all wells and mixedthoroughly to dissolve the dark blue formazan crystals. After anovernight incubation at 37° C., the optical densities at 540 nm weremeasured using a 96-well multiscanner autoreader, with thesolubilization buffer serving as blank. All assays were run intriplicate and results were expressed as IC₅₀ values. The IC₅₀ wasdefined as the concentration required for 50% reduction of the opticaldensity in each test, and was calculated as: (A₅₄₀ of drug-treatedwells—A₅₄₀ of control wells)/A₅₄₀ of drug-free wells×100.

Adhesion Assays

In vitro adhesion assays, Narla R K, Liu X P, Klis D, Uckun F M.Inhibition of human glioblastoma cell adhesion and invasion by4-(4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131) and4-(3′-bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline(WHI-P154). Clin Cancer Res 4:2463-71, 1998, were performed to (a) studythe baseline adhesive properties of U373 glioblastoma and TERA-2testicular cancer cell lines and (b) evaluate the effects of VDC andVDSeCN derivatives on the adhesive properties of U373 glioblastoma andTERA-2 testicular cancer cells. The plates for the adhesion assays wereprecoated with the extracellular matrix proteins laminin, fibronectin ortype IV collagen (each at a final concentration of 1 μg/ml in PBS)overnight at 4° C. and dried. On the day of the experiment, the wellswere rehydrated and blocked with 10% bovine serum albumin in PBS for 1hr at room temperature and used for the adhesion assays, as describedbelow. To study the effects of VDC and VDSeCN on cancer cell adhesion,exponentially growing cells in DMEM were incubated with these compoundsat concentrations ranging from 1 μM to 10 μM for 16 hr in a humidified5% CO₂ atmosphere. DMSO (0.1%) was included as a vehicle control. Aftertreatment, cells were detached from the flasks with 0.05% trypsin (LifeTechnologies) resuspended in DMEM, incubated at 37° C. for 2 hr to allowthem to recover from the trypsinization stress and examined for theirability to adhere to plates precoated with ECM proteins. Cells werecentrifuged, washed twice with serum-free DMEM, counted and resuspendedin serum-free DMEM to a final concentration of 2.5×10⁵ cells/ml. Onehundred μl of the cell suspension containing 2.5×10⁴ cells were added toeach well and cells were allowed to adhere for 1 hr at 37° C. in ahumidified 5% CO₂ atmosphere. The non-adherent cells were removed bygently washing the cells with PBS and then the adherent fraction wasquantitated using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays as described above. The adherent fractionsof cells treated with VDC or VDSeCN were compared to those of DMSOtreated control cells and the percent inhibition of adhesion wasdetermined using the formula: % Inhibition=100×(1-Adherent Fraction ofDrug-Treated Cells/Adherent Fraction of Control Cells). Each treatmentcondition was evaluated in duplicate in 3 independent experiments. TheIC₅₀ values were calculated by non-linear regression analysis using anGraphpad Prism software version 2.0 (Graphpad Software, Inc., San Diego,Calif.).

Cytotoxicity of Oxovanadium Compounds Against Human Cancer Cell Lines

A series oxovanadium (IV) complexes (Table 6) were prepared, includingeight phenanthroline (phen)-linked [VO(phen), VO(phen)₂, VO(Me₂-phen),VO(Me₂-phen)₂, VO(Cl-phen), VO(Cl-phen)₂, VO(NO₂-phen), VO(NO₂-phen)₂]and six bipyridyl (bipy)-linked [VO(bipy), VO(bipy)₂, VO(Me₂-bipy),VO(Me₂-bipy)₂, two bipyramide VO, and two bipyramide VO₂, and oneacetophenone (acph)-linked [VO(Br,OH-acph)₂] and tested their cytotoxicactivity against 14 different human cancer cell lines, including theB-lineage ALL cell line NALM-6, T-lineage ALL cell line MOLT-3, AML cellline HL-60, multiple myeloma cell lines ARH-77, U266BL, and HS-SULTAN,Hodgkin's lymphoma cell line HS445, and the testicular cancer cell lines833K, 64 cp5, TERA-2, and NTD1, prostate cancer cell line PC3, breastcancer cell line BT-20, and glioblastoma cell line U373 using MTT assaysand/or confocal laser scanning microscopy. Each compound was testedside-by-side at eight different concentrations in the range of 0.1-250μM.

Each of the 15 oxovanadium complexes exhibited significant cytotoxicityagainst several of the cancer cell lines in a concentration-dependentfashion (Table 7, FIG. 1). FIG. 1 shows the concentration-dependentMTT-based cytotoxicity curves of 12 representative oxovanadium (IV)compounds against NALM-6 leukemia cells. The cytotoxic activity of theoxovanadium(IV) complexes was strongly dependent on the type ofcoordinated heteroligands. When compared with diaqua mono-chelatedcomplexes, the butterfly structure oxovanadium complexes stabilized with5-membered bis-chelated ligands of phenanthroline or bipyridyl showedsuperior cytotoxic activity against cancer cells. The mono-chelated[VO(Me₂-phen)=compound 28] as well as bis-chelated-1,10-phenantrolinecomplexes [VO(Me₂-phen)₂=compound 29] were the most potent oxovanadiumcompounds and killed each of the 7 cell lines examined at low micromolarconcentrations (Table 7). Notably, the dimethyl substitution of thephenanthroline rings is believed to be significant for the anti-canceractivity of both compounds (29) [=VO(Me₂-phen)₂] and (28) [VO(Me₂-phen)]because unsubstituted bis-chelated and mono-chelated 1,10-phenanthrolineoxovanadium (IV) complexes [VO(phen)=compound 276 or VO(phen)₂=compound27] were less active. Addition of a chloro or nitro group to the1,10-phenanthroline complexes did not significantly improve thecytotoxic activity of the unsubstituted oxovanadium (IV) complexes(Table 7). Irrespective of the ligands, bis-chelated phenanthrolinecontaining compounds showed better activity than the mono-chelatedphenanthroline containing complexes. The marked differences in thecytotoxic activity of oxovanadium (IV) complexes containing differentheterocyclic ancillary ligands suggest that the cytotoxic activity ofthese compounds is determined by the identity of the 5-memberedbidentate ligands as well as the nature of the substitutents on theheterocyclic aromatic rings. The ability of oxovanadium compounds toinhibit the in vitro clonogenic growth of NALM-6 leukemia cells was alsoinvestigated. As detailed in Table 7, compounds 28 and 29 were the mostpotent compounds against clonogenic NALM-6 cells and completelyabrogated in vitro colony formation at concentrations <1 μM.

Oxovanadium (IV) Compounds Induce Apoptosis in Human Cancer Cells

In order to determine if the cytotoxicity of the oxovanadium compoundsis associated with apoptotic cell death, 64cp5 and 833-K testicularcancer cells were cultured with the oxovanadium compounds (50 μM) for 24h and then subjected to flow cytometric analysis for dUTP incorporationby the TdT-mediated TUNEL assay. FIG. 2A depicts the two-color flowcytometric contour plots of cells from representative TUNEL assays.Control 64cp5 and 833-K cells were treated for 24 hours at 37° C. with0.1% DMSO whereas test cells were treated for 24 hours at 37° C. with anoxovanadium compound at 50 μM final concentration. The TdT-dependentincorporation of FITC-dUTP was dramatically increased in cells treatedwith the oxovanadium compounds as a result of abundance of free3′-hydroxyl DNA ends created by endonuclease-mediated DNA fragmentation.Among the 15 vanadocenes evaluated by the flow cytometric TUNEL assay, 8caused a marked increase in TUNEL-positive nuclei ranging from 50.8 % to76.1 % for 64cp5 cells and 63.5% to 82.2% for 833-K cells respectively(FIG. 2B). Apoptosis after treatment with oxovanadium compounds was alsoevident from the concentration-dependent emergence of a hypodiploid(<2N) peak in the DNA histograms of PI-stained cells, which wasaccompanied by nonselective loss of G0/1, S, and G2M phase cells (FIG.2). Similar results were obtained with NALM-6 leukemia and HS-SULTANmultiple myeloma cells (Table 7, FIG. 3). As evidenced by the confocallaser scanning microscopy images depicted in FIG. 5, VO(Me₂-phen)₂(compound 29) and VO(Me₂-phen) (compound 28) treated [but notVO(Cl-phen)₂(=compound 31)-treated] leukemic NALM-6 and HS-SULTAN cellsexamined for FITC-conjugated dUTP incorporation (green fluorescence) andpropdium iodide counterstaining (red fluorescence) showed many apoptoticyellow nuclei with superimposed green and red fluorescence at 48 hoursafter treatment. Immunofluorescence staining with anti-α-tubulinantibody and the nuclear dye toto-3 in combination with confocal laserscanning microscopy was used to examine the morphological features ofcancer cells treated with oxovanadium compounds. FIG. 4 depicts thetwo-color confocal microscopy images of BT-20 breast cancer, PC3prostate cancer, and U373 glioblastoma cells after treatment withoxovanadium compounds. Most of the oxovanadium-treated cells displayedthe characteristic morphologic features of apoptotic cell death,including an abnormal architecture with complete disruption ofmicrotubules, marked shrinkage, chromatin condensation, nuclearfragmentation, the appearance of typical apoptotic bodies and inabilityto adhere to the substratum.

In order to test whether oxovanadium compounds induce apoptosis byaltering the mitochondrial transmembrane potential, NALM-6 leukemiacells were exposed to compound (29) for apoptosis-associated changes inmitochondrial membrane potential (ΔΨm) and mitochondrial mass usingspecific fluorescent mitochondrial probes and multiparameter flowcytometry. To measure changes in ΔΨm, DiIC1 (which accumulates inenergized mitochondria) was used, whereas the mitochondrial mass wasdetermined by staining the cells with NAO, a fluorescent dye that bindsto the mitochondrial inner membrane independent of energetic state.Treatment of NALM-6 leukemia cells with compound 29 for 24 h-72 hincreased the number of depolarized mitochondria in a concentration- andtime-dependent fashion, as determined by flow cytometry using DiIC1(27-29) (FIG. 5A). As shown in FIG. 5A, the fraction of DiIC1-negativecells with depolarized mitochondria increased from 6.5% in vehicletreated control cells to 91.5% in cells treated with 1 μM compound 29for 48 hours. The average EC₅₀ values for compound (29) induceddepolarization of mitochondria, as measured by decreased DiIC1 stainingwere 3.6 μM, 0.3 μM and 0.08 μM for 24 hr 48 hr and 72 hr treatmentrespectively. The observed changes in ΔΨm were not due to loss inmitochondrial mass, as confirmed by a virtually identical stainingintensity of NAO in the treated and untreated NALM-6 cells (FIG. 5B). Tofurther confirm this relative change in ΔΨm, JC-1, a mitochondrial dye,which normally exists in solution as a monomer emitting greenfluorescence and assumes a dimeric configuration emitting redfluorescence in a reaction driven by mitochondrial transmembranepotential was used. Thus, the use of JC-1 allows simultaneous analysisof mitochondrial mass (green fluorescence) and mitochondrialtransmembrane potential (red/orange fluorescence). After treatment ofNALM-6 cells with compound 29 at increasing concentrations ranging from500 nM to 1 μM and with increasing duration of exposure of 24 h or 48 h,a progressive dissociation between ΔΨm and mitochondrial mass wasobserved, with decrement in JC-1 red/orange fluorescence without asignificant corresponding drop in JC-1 green fluorescence. The fractionof JC-1 red/orange fluorescence-positive cells decreased from 98.6% invehicle-treated control cells to 56.1% in cells treated with 500 nM ofcompound 29 for 48 hr and 8.4% in cells treated with 500 nM of compound29 for 72 hr. The average EC₅₀ for compound 29-induced depolarization ofmitochondria, as measured by JC-1 red/orange fluorescence were 4.2 μMfor the 24 hr treatment and 0.5 μM for the 48 hr treatment. Theseresults collectively demonstrate that compound 29 causes a significantdecrease in mitochondrial transmembrane potential in NALM-6 humanleukemia cells.

The results indicate that oxovanadium (IV) complexes with1,10-phenanthroline, 2,2′-bipyridyl, or 5′-bromo-2′-hydroxyacetophenoneand their derivatives linked to vanadium(IV) via nitrogen or oxygenatoms have potent anti cancer activity against human cancer cells. Theorder of efficacy for the 15 oxovanadium (IV) complexes against NALM-6leukemia cells as follows:VO(Me₂-phen)₂>VO(NO₂-phen)>VO(Me₂-phen)>VO(phen)₂,>VO(Cl-phen)₂>VO(phen)>VO(Cl-phen)>VO(Me₂-bipy)₂>VO(NO₂-phen)>VO(Me₂-bipy)>VObipym>VObipym₂>VO(bipy)>VO(bipy)₂>VO(Br,OH-acph)₂.

Apoptosis induction of cytotines of the oxovanadium compounds of thepresent invention extend to human sperm. Therefore, these compounds arealso useful as sperm or birth control.

TABLE 7 Cytotoxic of Activity Oxovanadium (IV) Compounds AgainstLeukemia, Hodgkin's Lymphoma and Multiple Myeloma Cells NALM-6 MOLT-3HS445 HL-60 U266BL ARH77 HS-SULTAN Compound IC₅₀ (μM) IC₅₀ (μM) IC₅₀(μM) IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) 26  3.4 ± 0.2 2.7 ± 0.2 6.3± 1.8 3.2 ± 1.1  3.5 ± 0.5 15.6 ± 3.2 11.2 ± 2.2 27 0.97 ± 0.1  1.4 ±0.04 5.8 ± 1.2 4.6 ± 1.4  2.2 ± 0.6  3.3 ± 0.8  4.4 ± 0.8 28 0.78 ± 0.11.37 ± 0.07 3.3 ± 1.4 6.2 ± 2.3  1.3 ± 0.1  8.1 ± 1.6  5.1 ± 1.02 29 0.2 ± 0.03 0.19 ± 0.01  0.5 ± 0.08 0.98 ± 0.1   0.5 ± 0.02 0.81 ± 0.9 0.8 ± 0.05 30  3.6 ± 0.07  3.4 ± 0.03 7.5 ± 2.5 15.3 ± 4.9   8.5 ± 1.230.3 ± 5.6  9.9 ± 5.4 31  1.6 ± 0.03 2.1 ± 0.2 5.2 ± 1.1 5.3 ± 1.8  3.7± 1.1 10.5 ± 3.8  5.8 ± 0.6 32  4.1 ± 0.4 2.3 ± 0.3 9.1 ± 2.5 4.9 ± 1.218.1 ± 5.1  5.4 ± 1.5 14.5 ± 2.5 33  0.7 ± 0.01  1.4 ± 0.06 2.3 ± 0.62.6 ± 0.4  7.2 ± 1.1  5.7 ± 1.3 13.8 ± 3.6 34 14.9 ± 0.6 13.1 ± 1.1 41.7 ± 5.4  >100 30.4 ± 7.8 >100 62.8 ± 2.3 35 15.5 ± 3.8 14.2 ± 3.8 21.6 ± 4.3  31.4 ± 6.9  32.1 ± 5.2 26.8 ± 5.4 35.4 ± 3.3 36  8.5 ± 0.58.2 ± 0.5 27.8 ± 3.4  38.6 ± 4.5  38.4 ± 3.2 >100 65.1 ± 6.1 37  3.9 ±0.3 4.8 ± 0.3 12.6 ± 3.3  28.4 ± 3.8  13.3 ± 5.1 58.3 ± 6.8 11.4 ± 2.238 12.1 ± 1.6 27.7 ± 2.5  96.5 ± 8.6  >100 91.2 ± 9.9 >100 >100 39 12.2± 2.3 35.1 ± 4.7  98.4 ± 9.1  >100 99.1 ± 8.1 >100 >100 40 17.4 ± 0.941.8 ± 3.9  96.5 ± 6.4  >100 98.1 ± 6.7 >100 78.4 ± 5.8 Cells weretreated with various concentrations ranging from 0.1 μM to 100 μM ofoxovanadium (IV) complexes for 48 hr and the cell survival was measuredwith MTT assays and EC₅₀s were calculated with non-linear regressionanalysis.

TABLE 8 Cytotoxic Activity of Oxovanadium (IV) Compounds AgainstTesticular cancer, Brain tumor, Breast Cancer and Prostate Cancer cellsCom- TERA- pound 833-K 64cp5 2 NTD1 U373 BT20 PC3 26 12.8 8.5 >100 >1007.7 6.6 11.2 27 7.5 18.5 37.6 >100 6.8 7.2 10.8 28 6 15.2 57.6 12.8 2.22.1 5.8 29 0.85 0.75 19.5 10.8 1.8 1.5 1.7 30 12.8 10.9 18.1 >100 22.418.2 12.4 31 4.6 >100 24.2 50.1 6.3 5.4 4.6 32 11.5 25.4 7.2 20.2 20.513.7 12.9 33 1.1 5.9 2.6 9.1 7.2 5.6 6.1 34 >100 >100 >100 >100 58.451.2 55.8 35 96.2 >100 >100 >100 >100 70.8 56.236 >100 >100 >100 >100 >100 >100 N.D. 37 34.9 >100 >100 >100 25.6 15.7N.D. 38 >100 >100 74.3 >100 >100 >100 N.D.39 >100 >100 >100 >100 >100 >100 N.D. 40 >100 >100 35.4 >100 >100 >100N.D.

EXAMPLE 3

The following illustrate representative pharmaceutical dosage forms,containing a compound of formula I (‘Compound X’), for therapeutic orprophylactic use in humans.

(I) Tablet 1 mg/tablet ‘Compound X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0 (ii) Tablet 2 mg/tablet ‘Compound X’ 20.0Microcrystalline cellulose 410.0 Starch 50.0 Sodium starch glycolate15.0 Magnesium stearate 5.0 500.0 (iii) Capsule mg/capsule ‘Compound X’10.0 Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelatinized starch120.0 Magnesium stearate 3.0 600.0 (iv) Injection 1 (1 mg/ml) mg/ml‘Compound X’ (free acid form) 1.0 Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodium chloride 4.5 1.0 N Sodiumhydroxide solution q.s. (pH adjustment to 7.0-7.5) Water for injectionq.s. ad 1 mL (v) Injection 2 (10 mg/ml) mg/ml ‘Compound X’ (free acidform) 10.0  Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1Polyethylene glycol 400 200.0  01 N Sodium hydroxide solution q.s. (pHadjustment to 7.0-7.5) Water for injection q.s. ad 1 mL (vi) Aerosolmg/can ‘Compound X’ 20.0 Oleic acid 10.0 Trichloromonofluoromethane5,000.0 Dichlorodifluoromethane 10,000.0 Dichlorotetrafluoroethane5,000.0

The above formulations may be obtained by conventional procedures wellknown in the pharmaceutical art.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A compound of formula IV:

wherein R⁵ is hydrogen; R⁴ and R⁶ are each independently (C1-C3)alkyl,halogen, (C1-C3)alkoxy, halo (C1-C3) alkyl, cyano, (C2-C6)alkanoyloxy ornitro; X⁴ and X⁵ are each independently OH₂ or no ligand is present onX⁵ or taken together X⁴ and X⁵ form a N,N-bidentate ligand selected fromthe group consisting of N,N-bipyridine, N,N-bipyrimidine, andN,N-phenanthroline, wherein the N,N-bidentate ligand can be substitutedwith up to two groups that are independently (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo (C1-C3)alkyl, cyano, (C2-C6)alkanoyloxy or nitro; Yis OH₂ or OSO₃; or a pharmaceutically acceptable salt thereof.
 2. Thecompound of claim 1, wherein the compound is [VO(Me₂-Phen)(H₂O)₂](SO₄)or [VO(SO₄)(Me₂-Phen)₂].
 3. A method for treating leukemia, Hodgkins'slymphoma, multiple myeloma, testicular cancer, brain tumor, breastcancer, or prostate cancer in a mammal comprising administering to themammal in need of such treatment an effective amount of a vanadium (IV)compound, wherein the vanadium (IV) compound is a compound of formulaIV:

wherein R⁵ is hydrogen; R⁴ and R⁶ are each independently (C1-C3)alkyl,halogen, (C1-C3)alkoxy, halo (C1-C3)alkyl, cyano, (C2-C6)alkanoyloxy ornitro; X⁴ and X⁵ are each independently OH₂ or no ligand is present onX⁵ or taken together X⁴ and X⁵ form a N,N-bidentate ligand selected fromthe group consisting of N,N-bipyridine, N,N-bipyrimidine, andN,N-phenanthroline, wherein the N,N-bidentate ligand can be substitutedwith up to two groups that are independently (C1-C3) alkyl, halogen,(C1-C3)alkoxy, halo (C1-C3)alkyl, cyano, (C2-C6) alkanoyloxy or nitro;and Y is OH₂ or OSO₃; or a pharmaceutically acceptable salt thereof. 4.The method of claim 3 wherein the compound is [VO(Me₂-Phen)(H₂O)₂](SO₄)or [VO(SO₄)(Me₂-Phen)₂].
 5. A pharmaceutical composition comprising acompound of formula IV:

wherein R⁵ is hydrogen; R⁴ and R⁶ are each independently (C1-C3)alkyl,halogen, (C1-C3)alkoxy, halo (C1-C3)alkyl, cyano, (C2-C6)alkanoyloxy ornitro; X⁴ and X⁵ are each independently OH₂ or no ligand is present onX⁵ or taken together X⁴ and X⁵ form a N,N-bidentate ligand selected fromthe group consisting of N,N-bipyridine, N,N-bipyrimidine, andN,N-phenanthroline, wherein the N,N-bidentate ligand can be substitutedwith up to two groups that are independently (C1-C3)alkyl, halogen,(C1-C3)alkoxy, halo (C1-C3)alkyl, cyano, (C2-C6)alkanoyloxy or nitro;and Y is OH₂ or OSO₃; or a pharmaceutically acceptable salt thereof; anda pharmaceutically acceptable carrier.
 6. The pharmaceutical compositionof claim 5 wherein the compound of formula IV is[VO(Me₂-Phen)(H₂O)₂](SO₄) or [VO(SO₄)(Me₂-Phen)₂].
 7. A compound offormula IV:

wherein R⁴ and R⁶ are hydrogen; R⁵ is (C2-C3)alkyl, halogen,(C1-C3)alkoxy, halo (C1-C3)alkyl, cyano, (C2-C6)alkanoyloxy or nitro; X⁴and X⁵ are each independently OH2 or no ligand is present on X⁵ or takentogether X⁴ and X⁵ are a N,N-bidentate ligand selected from the groupconsisting of N,N-bipyridine, N,N-bipyrimidine, and N,N-phenanthroline,wherein the N,N-bidentate ligand can be substituted with up to twogroups that are independently (C1-C3)alkyl, halogen, (C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C2-C6)alkanoyloxy or nitro; and Y is OH₂ or OSO₃;or a pharmaceutically acceptable salt thereof.
 8. The compound of claim7 wherein the compound is [VO(Cl-Phen)(H₂O)₂](SO₄), [VO(SO₄)(Cl-Phen)₂],[VO(NO₂-Phen)(H₂O)₂](SO₄), or [VO(SO₄)(NO₂-Phen)₂].
 9. A method fortreating leukemia, Hodgkins's lymphoma, multiple myeloma, testicularcancer, brain tumor, breast cancer, or prostate cancer in a mammalcomprising administering to the mammal in need of such treatment aneffective amount of a vanadium (IV) compound, wherein the vanadium (IV)compound is a compound of formula IV:

wherein R⁴ and R⁶ are hydrogen; R⁵ is (C2-C3)alkyl, halogen,(C1-C3)alkoxy, halo (C1-C3)alkyl, cyano, (C2-C6)alkanoyloxy or nitro; X⁴and X⁵ are each independently OH₂ or no ligand is present on X⁵ or takentogether X⁴ and X⁵ are a N,N-bidentate ligand selected from the groupconsisting of N,N-bipyridine, N,N-bipyrimidine, and N,N-phenanthroline,wherein the N,N-bidentate ligand can be substituted with up to twogroups that are independently (C1-C3)alkyl, halogen, (C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C2-C6)alkanoyloxy or nitro; and Y is OH₂ or OSO₃;or a pharmaceutically acceptable salt thereof.
 10. The method of claim9, wherein the compound is [VO(Cl-Phen)(H₂O)₂](SO₄),[VO(SO₄)(Cl-Phen)₂], [VO(NO₂-Phen)(H₂O)₂](SO₄), or [VO(SO₄)(NO₂-Phen)₂].11. A pharmaceutical composition comprising a compound of formula IV:

wherein R⁴ and R⁶ are hydrogen; R⁵ is (C2-C3)alkyl, halogen,(C1-C3)alkoxy, halo (C1-C3)alkyl, cyano, (C2-C6)alkanoyloxy or nitro; X⁴and X⁵ are each independently OH₂ or no ligand is present on X⁵ or takentogether X⁴ and X⁵ are a N,N-bidentate ligand selected from the groupconsisting of N,N-bipyridine, N,N-bipyrimidine, and N,N-phenanthroline,wherein the N,N-bidentate ligand can be substituted with up to twogroups that are independently (C1-C3)alkyl, halogen, (C1-C3)alkoxy, halo(C1-C3)alkyl, cyano, (C2-C6)alkanoyloxy or nitro; and Y is OH2 or OSO₃;or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.
 12. The pharmaceutical composition of claim 11,wherein the compound is [VO(Cl-Phen)(H₂O)₂](SO₄), [VO(SO₄)(Cl-Phen)₂],[VO(NO₂-Phen)(H₂O)₂](SO₄), or [VO(SO₄)(NO₂-Phen)₂].